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Chronic obstructive pulmonary disease (COPD) is associated with age and smoking, but other determinants of the disease are incompletely understood. Clonal hematopoiesis of indeterminate potential (CHIP) is a common, age-related state in which somatic mutations in clonal blood populations induce aberrant inflammatory responses. Patients with CHIP have an elevated risk for cardiovascular disease, but the association of CHIP with COPD remains unclear. We analyzed whole-genome sequencing and whole-exome sequencing data to detect CHIP in 48 835 patients, of whom 8444 had moderate to very severe COPD, from four separate cohorts with COPD phenotyping and smoking history. We measured emphysema in murine models in which Tet2 was deleted in hematopoietic cells. In the COPDGene cohort, individuals with CHIP had risks of moderate-to-severe, severe, or very severe COPD that were 1.6 (adjusted 95% confidence interval [CI], 1.1-2.2) and 2.2 (adjusted 95% CI, 1.5-3.2) times greater than those for noncarriers. These findings were consistently observed in three additional cohorts and meta-analyses of all patients. CHIP was also associated with decreased FEV1% predicted in the COPDGene cohort (mean between-group differences, −5.7%; adjusted 95% CI, −8.8% to −2.6%), a finding replicated in additional cohorts. Smoke exposure was associated with a small but significant increased risk of having CHIP (odds ratio, 1.03 per 10 pack-years; 95% CI, 1.01-1.05 per 10 pack-years) in the meta-analysis of all patients. Inactivation of Tet2 in mouse hematopoietic cells exacerbated the development of emphysema and inflammation in models of cigarette smoke exposure. Somatic mutations in blood cells are associated with the development and severity of COPD, independent of age and cumulative smoke exposure.

The leukocyte adhesion cascade is important in chronic lymphocytic leukemia (CLL), as it controls migration of malignant cells into the pro-survival lymph node microenvironment. Circulating trisomy 12 CLL cells have increased expression of the integrins CD11a and CD49d, as well as CD38, but the tissue expression of these and other molecules, and the functional and clinical sequelae of these changes have not been described. Here, we demonstrate that circulating trisomy 12 CLL cells also have increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and the adhesion molecule CD323. Notably, there was reduced expression of CD11a, CD11b, and CD18 in trisomy 12 cases with NOTCH1 mutations compared with wild type. Trisomy 12 cells also exhibit upregulation of intracellular integrin signaling molecules CALDAG-GEFI, RAP1B, and Ras-related protein ligand, resulting in enhanced very late antigen-4 [VLA-4] directed adhesion and motility. CD38 expression in CLL has prognostic significance, but the increased CD38 expression in trisomy 12 CLL cells must be taken into account in this subgroup, and the threshold of CD38 positivity should be raised to 40% for this marker to retain its prognostic value. In conclusion, trisomy 12 CLL cells exhibit functional upregulation of integrin signaling, with β2-integrin expression being modulated by NOTCH1 mutation status.

Background: Patients (pts) with MDS or AML who relapse after allogeneic transplantation (allo-HCT) have a very poor prognosis. Hypomethylating agents (HMA) and checkpoint blockade with the anti-CTLA4 blocking antibody ipilimumab (IPI) have each induced responses with acceptable toxicity in AML pts who relapse after allo-HCT. We hypothesized that adding decitabine (DAC) would improve response compared with IPI alone by activating and promoting T cell-mediated anti-leukemic immune reactivity. We are conducting a multicenter phase I study (CTEP 10026) of DAC plus IPI in pts with R/R MDS/AML in both post allo-HCT and transplant-naïve settings to assess safety and estimate efficacy. Methods: The primary objective is to determine the maximum tolerated dose (MTD) or RP2D of combination DAC + IPI in pts with R/R MDS/AML who are post allo-HCT (Arm A) or transplant-naïve (Arm B). Cohorts of 3-6 are sequentially enrolled in 3 dose levels (DL) of IPI using a 3+3 design with expansion in each arm; DLs 0-2 are 3, 5 and 10 mg/kg, respectively. Eligibility for both arms: relapsed AML (extramedullary or ≥ 5% blasts) or R/R MDS (≥ 5% blasts) or unfit elderly AML; Arm A only: ≥ 2 wks off systemic immunosuppressive (IS) therapy, T cell chimerism ≥ 20%, and no prior acute GVHD ≥ gr III. DLT is defined as ≥ gr 3 non-heme, ≥ gr 3 acute GVHD or ≥ gr 3 steroid-refractory immune-related adverse events (AEs) occurring within 8 weeks from first IPI dose. Epigenetic priming with DAC lead-in cycle 0 was followed by combination cycles of DAC + IPI. DAC is given at 20 mg/m2 days 1-5 q 28 days. IPI is given on day 1 of cycles 1-4 and every other cycle in cycles 5-12. Pts who discontinued study either in cycle 0 or DLT period without IPI-toxicity were replaced. Arm A opened after safety was confirmed at DL0 in Arm B. Results: As of June 9, 2019, 26 pts (15M, 11 F) have enrolled in this on-going trial. Of the 12 pts (11 AML and 1 MDS) enrolled in Arm A (post allo-HCT), median age was 66.5 (range 29-74) and 9 had previously received HMA. 7 of 8 pts in DL0 (1 progressed in cycle 0) and 3 of 4 pts in DL1 (1 died from pneumonia in cycle 0) received DAC + IPI. DL0 was expanded to 6 to confirm safety without DLT. Median treatment duration after first IPI dose was 5 cycles (range 1-7); 4 pts continue on trial. Common AEs were gr 1-2 dyspnea (n=4), gr 1-3 fatigue (n=4), and gr 1-2 fever (n=4). Gr 3 AEs were febrile neutropenia (n=2), pneumonia (n=1), and candidemia (n=1). Gr 1 immune-related dermatitis (n=1) reversed with steroids. Acute GVHD was not observed. Moderate-severe chronic GVHD was noted in 2 pts mainly involving skin, which was responsive to photopheresis and oral IS. Though 1 CR and 1 marrow CR have been observed at DL0, dose-escalation up to DL2 is on-going to determine MTD. Of the 14 pts (11 AML and 3 MDS) enrolled in Arm B (transplant naïve), median age was 75.5 (range 34-82) and 9 had previously received HMA. 4 of 6 pts in DL0 (1 progressed and 1 withdrew in cycle 0), 3 of 5 pts in DL1 (2 withdrew in cycle 0) and 3 of 3 pts in DL2 received DAC + IPI. Median treatment duration after first IPI dose was 4 cycles (range 1-8); 3 pts remain on study. Common AEs were gr 1-3 fatigue (n=9), gr 1-2 anorexia (n=5), and gr 3 febrile neutropenia (n=8). Immune-related gr 2 colitis (n=1) and gr 2/3 (n=4) dermatitis were all steroid-responsive. Of the 10 pts who received at least one IPI dose, 5 (50%) achieved an objective response including 3 CR, 1 CRi and 1 PR. All responses were observed in AML pts, including 1 with only skin involved. Expansion to confirm MTD is underway. No treatment-related deaths or DLTs were observed in either Arm. Reasons for discontinuation after IPI: progression (n=9), proceeding to allo-HCT or DLI (n=2), withdrawal (n=1), stroke due to underlying atrial fibrillation (n=1) and disseminated nocardiosis (n=1). In both Arms, multiplex immunofluorescence (MIF) staining of BM biopsies revealed a higher density of CD3+CD4+ cells after 4 cycles of DAC + IPI in 4 responders (R) compared to 4 non-responders (NR) (p=0.0433). Longitudinal MIF IHC in an Arm B responder identified the increasing presence of a tumor immune infiltrate composed of CD3+CD8+GZMB+ T cells prior to achieving CR (Fig 1). Conclusions: Combination DAC + IPI is tolerable and has encouraging clinical activity in post allo-HCT and transplant naïve pts with R/R MDS/AML. Ongoing studies focus on comparing the immunologic and genetic characteristics of the tumor immune infiltrate in each cohort to understand the contribution of alloimmunity to treatment response. Disclosures Garcia: Abbvie: Research Funding; Genentech: Research Funding. Keng:agios: Membership on an entity's Board of Directors or advisory committees. Brunner:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Research Funding. Khaled:Omeros: Consultancy; Alexion: Consultancy, Speakers Bureau; Daiichi Sankyo: Other: Travel support. Steensma:H3 Biosciences: Other: Research funding to institution, not investigator.; Arrowhead: Equity Ownership; Onconova: Consultancy; Stemline: Consultancy; Aprea: Research Funding; Pfizer: Consultancy; Summer Road: Consultancy; Astex: Consultancy. Winer:Jazz Pharmaceuticals, Pfizer: Consultancy. Cutler:Omeros: Consultancy; Kadmon: Consultancy; BiolineRx: Other: DSMB; Cellect: Other: DSMB; Kalytera: Other: DSMB; ElsaLys: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Fate Therapeutics: Consultancy; Incyte: Consultancy; Jazz: Consultancy; BMS: Consultancy. Ho:Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Neuberg:Madrigal Pharmaceuticals: Equity Ownership; Pharmacyclics: Research Funding; Celgene: Research Funding. Lindsley:Takeda Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Research Funding; Medlmmune: Research Funding. Galinsky:ABIM: Other: Member of specialty oncology board; Merus Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; AbbVie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Ritz:TScan Therapeutics: Consultancy; LifeVault Bio: Consultancy; Kite Pharma: Research Funding; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; Avrobio: Consultancy; Celgene: Consultancy; Merck: Research Funding; Equillium: Research Funding; Aleta Biotherapeutics: Consultancy. Davids:AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding; Research to Practice: Honoraria. Wu:Pharmacyclics: Research Funding; Neon Therapeutics: Other: Member, Advisory Board. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. DeAngelo:Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Abbvie: Research Funding; Glycomimetics: Research Funding; Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy. Soiffer:Jazz: Consultancy; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Kiadis: Other: supervisory board; Mana therapeutic: Consultancy; Cugene: Consultancy. OffLabel Disclosure: Combination of ipilimumab and decitabine for MDS/AML treatment for patients who are post-transplant or transplant naive

Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the transformation of CLL to an aggressive lymphoma, or Richter's transformation (RT), remains a clinical challenge, as it responds poorly to standard therapies and shortens survival. Recent studies demonstrate that RT, but not underlying CLL, responds to PD-1 checkpoint blockade (CPB) with an overall response rate of 43-65%. Given the central role of T cells in anti-tumor immunity, we hypothesized that differences in T cell populations underlie response and resistance to CPB in RT. We focused on a discovery cohort of 6 patients with RT (4 responders, 2 non-responders) and 2 patients with relapsed/refractory CLL enrolled on a study in which patients were initiated with anti-PD1 therapy (nivolumab 3 mg/kg every 2 weeks), with subsequent concurrent ibrutinib (420 mg daily)(NCT 02420912). We examined a total of 15 serial study marrow specimens collected at treatment initiation and 3 month response evaluation, as well as 2 healthy marrow donors. To systematically discover the T cell populations and states associated with CPB response in RT, we performed single-cell RNA-sequencing (scRNA-seq, 10x Genomics) of non-lymphoma (CD5-CD19-) cells isolated by flow cytometry from marrow samples. A total of 60,727 T and NK cells were captured with average detection of 1001 genes/cell. Using the novel joint clustering approach Conos, 11 transcriptionally distinct clusters of lymphocytes were identified. We first contrasted baseline RT/CLL with normal marrow and observed differences across T cell populations, which we confirmed through the examination of publicly available marrow scRNA-seq data from 28 healthy donors. Compared to normal marrow, RT/CLL marrow was enriched for cytotoxic populations, including both CD8 effector/effector memory (E/EM) (p=0.001, t-test) and cytotoxic CD4 (p=0.001) T cells as well as for cells expressing multiple exhaustion markers, including PDCD1, LAG3 and TIGIT (p=0.001). In contrast, normal marrow contained increased T cells with a naïve-like phenotype (p=0.06). When we focused on the pre-treatment samples from RT patients, RT responders had a larger CD8 E/EM population (p=0.04) and fewer T regulatory cells (p=0.006, t-test) than RT non-responders. Using DESeq2 to compare clusters from all samples, we evaluated if there were differences in gene expression between RT responders and non-responders. CD8 E/EM T cells of RT non-responders showed increased expression of TOX, a recently uncovered master regulator of cell exhaustion (padj =0.00016), while this cell subtype in RT responders upregulated a contrasting program of activating transcription factors as well as the co-stimulatory gene CD226 (padj =0.04). As for CD4 T cells, RT responders revealed an enriched cytotoxic gene program compared to RT non-responders (padjPRF1 5.9 x 10-10, GZMH 6.0 x 10-6, NKG7 6.4 x 10-19). To investigate whether response to CPB therapy for RT was associated with changes in the T cell receptor (TCR) repertoire, and to obtain protein-level validation of transcriptional signatures, we performed single-cell TCR sequencing with paired gene and protein expression (10x Genomics) on pre- and post-therapy samples from a RT responder and a non-responder. Indeed, we confirmed our gene expression findings, including validation of cytotoxic CD4 T cells and the enrichment of CD226 protein in E/EM CD8 T cells in the RT responder. TCR clonal expansion was observed in the RT responder at baseline with persistence of enriched clonotypes following CPB, suggesting the presence of tumor-reactive T cell clones. In contrast, the RT non-responder displayed higher TCR diversity with enriched clonotypes showing increased exhaustion post-CPB (p In conclusion, we identified marrow T cell populations enriched in RT patients and described distinct T cell transcriptional programs that delineate RT responders from non-responders. We have thus discovered candidate gene biomarkers that may identify patients likely to respond to CPB therapies and uncovered a CD8 E/EM T cell population that is likely to underlie response to PD-1 CPB. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Neuberg:Celgene: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Pharmacyclics: Research Funding. Rodig:Bristol Myers Squib: Consultancy, Honoraria, Other: Travel Expenses, Speakers Bureau; Kite, a Gilead Company: Research Funding; Affirmed: Research Funding; Merck: Research Funding. Wierda:KITE pharma: Research Funding; Gilead Sciences: Research Funding; AbbVie: Research Funding; Acerta Pharma Inc: Research Funding; Genentech: Research Funding; GSK/Novartis: Research Funding; Pharmacyclics LLC: Research Funding; Sunesis: Research Funding; Miragen: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Cyclcel: Research Funding; Loxo Oncology Inc.: Research Funding; Janssen: Research Funding; Xencor: Research Funding; Juno Therapeutics: Research Funding. Jain:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Background: To investigate the role of genomics in determining response and resistance to front line treatment in MPN, we performed somatic mutational profiling of the DALIAH trial, a randomized controlled phase III trial of interferon versus hydroxyurea in newly diagnosed MPN patients. Methods: We performed genomic analyses on 202 pre-treatment primary MPN samples obtained from patients enrolled on the DALIAH trial (NCT01387763) and 135 samples obtained after 24 months of treatment. Genomic profiling comprised targeted next generation sequencing (NGS) of 100 genes, selected on the basis of their known or suspected involvement in the pathogenesis of myeloid malignancies, and 1609 informative single nucleotide polymorphisms (SNPs) on chromosome 9p. Clinicohematological response was determined by central review using ELN 2009 (ET, PV and pre-MF) and EUMNET 2005 (PMF) criteria. We evaluated the association of somatic mutations with clinical parameters and with attainment of clinicohematological complete response (CR) at 24 months. Results: Prior to treatment, 191 of 202 (95%) patients had somatic mutations, including 93% with canonical MPN phenotypic drivers: JAK2 (74%), CALR (14%), and MPL (5%). Among those with JAK2 mutations 37% had JAK2 copy-neutral loss of heterozygosity (JAK2 CN-LOH). Patients with PV were more likely to have JAK2 CN-LOH (p = 0.0001) as compared with patients with other MPN subtypes. At baseline, patients with JAK2 CN-LOH had significantly higher hemoglobin (p = 0.0001), higher white blood cell count (WBC, p = 0.002) and lower platelet count (p=0.0001) than patients without JAK2 CN-LOH. Mutations in TET2 (24%), DNMT3A (16%), and ASXL1 (10%) were the most frequent co-occurring non-MPN phenotypic driver mutations and they occurred across all MPN subtypes. In addition, 5% of patients had spliceosome gene mutations, and 6% had mutations in genes involved in RAS/MAPK signaling. Patients with TET2, DNMT3A or ASXL1 mutations were significantly older than patients without these mutations (p= 0.0001) and there was a significant association between the presence of a TET2, DNMT3A or ASXL1 mutation and prior stroke (p = 0.004). There were no other significant associations between somatic mutation status and baseline clinical characteristics. The probability of attaining clinicohematological CR at 24 months was independent of baseline somatic mutations. Among patients with JAK2 mutations who remained on interferon treatment at 24 months, those with CR had a greater reduction in mean variant allele fraction (VAF) (28% to 8%, p Among patients who remained on treatment for 2 years, 44 mutations in 35 patients were newly detected or expanded on serial sampling. DNMT3A mutations were the most frequently acquired, accounting for 41% of new mutations. ASXL1, TET2, PPM1D, TP53, IDH2, and CBL mutations were also recurrently acquired. 97% of patients who acquired new mutations were JAK2-mutant. Among those with acquired DNMT3A mutations, 85% were treated with interferon, and 23% had CR at 24 months. Among those that acquired non-DNMT3A mutations, 38% were treated with interferon and 47% had CR at 24 months. The VAF of newly detected mutations was low (median 1.5%), and half of the patients with newly acquired mutations had at least 50% reduction in JAK2V617F VAF, suggesting that new mutations could either have arisen independently or be subclonal to the dominant JAK2-mutant clone. Conclusions: Using sequential genomic analyses of a phase III clinical trial of interferon versus hydroxyurea in MPN patients, we found mutation-specific and treatment-specific patterns of response. We uncovered distinct patterns of response to interferon in JAK2-mutant MPN as compared with CALR-mutant MPN. We found that DNMT3A mutations were the most frequent acquired mutations at 24 months and that these were enriched in patients treated with interferon. In aggregate, these results provide insights into molecular response and resistance to interferon and inform the clinical use of interferon in MPN patients. Disclosures Hansen: Alexion: Research Funding. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Hasselbalch:Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board. Lindsley:Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Mullally:Janssen: Research Funding.

Background: Although daunorubicin/cytarabine ("3+7")-based chemotherapy (CT) regimens produce complete remission (CR) rates of approximately 70% in younger adults, about 30% require re-induction. Among those achieving CR, relapse after post-remission intensive CT or allogeneic transplant is common and limits cure. Venetoclax (VEN) is an orally available selective BCL-2 inhibitor that promotes mitochondrial-mediated apoptosis in myeloblasts during cytotoxic stress. VEN in combination with hypomethylating agents and low dose cytarabine in untreated unfit elderly AML patients (pts) elicits high response and led to drug approval in that setting. VEN in combination with standard '3+7" could lead to a higher rate of measurable residual disease (MRD)-negative CR, perhaps obviating the need for reinduction as well as limiting relapse after consolidation therapy without undue toxicity. Methods: The primary objective of this phase 1 study is to determine the maximum tolerated dose of VEN that can be given in combination with standard intensive induction (ind) and consolidation CT in previously untreated AML pts. Pt eligibility includes no prior treatment (rx) for AML, FLT3 wild type, non-inv16 or t(8; 21) AML, either de novo or secondary. Pts must not require strong CYP3A inhibitors/inducers, have normal organ function, have WBC=18 (study amended to include only those age 18-60 after the first 6 pts were treated). VEN is given during ind on days 1-11 in cohorts of 200, 400, and 600 mg/d/ (after 4 d ramp-up) with daunorubicin 60 mg/m2/d IVP days 2-4 and cytarabine 200 mg/m2/d by IVCI days 2-8. A day 15 marrow determines the need for a second ind consisting of the same doses of VEN, daunorubicin and cytarabine given on days 1-8, 2-3, and 2-6, respectively. VEN escalation is carried out in 3+3 fashion. Once the MTD for VEN during ind is determined pts will be treated at that dose during ind and remitting pts given escalating doses (cohorts of 200, 400, 600 mg) of VEN d1-8 in a 3+3 fashion (beginning at one dose level below the MTD (or 100 mg if 200 mg is determined to the ind MTD)) with a single cycle of cytarabine 3g/m2 over 3h q 12 h on days 1, 3, and 5. A 10 pt confirmation cohort will be accrued at the MTD. Dose-limiting toxicity (DLT) is defined as any death, delayed neutrophil recovery (failure to achieve absolute neutrophil count (ANC) ≥ 500/mL by day 42 in pts without residual AML), or any gr 4 non-heme non-resolving toxicity not clearly due to CT alone. A marrow exam, including an assessment of MRD by multiparameter flow cytometry with a sensitivity of 0.02%, is carried out at the time of count recovery during induction but no later than day 42. Correlative studies include flow cytometry-based BH3 profiling (Vo TT, Cell, 2012) on blasts obtained at enrollment and just prior to chemo and CyTof to characterize BCL-2-associated proteins. Results: The first pt treated (58 yo F with normal karyotype, and NGS panel showing a DNMT3A mutation, though subsequent PCR-based assay disclosed a FLT3 ITD) developed gr 4 DIC after the first dose of VEN 50 mg (required factor replacement for elevated INR, but had no bleeding). VEN was stopped and she achieved an MRD negative CR with standard ind, but the first cohort was expanded to 6 pts. 4 of the 5 additional pts achieved a CR, but a 73 year old man died of nadir sepsis at d 14. Given 2/6 DLT events, VEN 200 was considered unacceptable by protocol design. An amendment allowed further accrual at the starting dose of VEN 200 mg but restricted accrual to age 18-60. All 3 pts on the expanded cohort have responded, and none experienced a DLT, so accrual to the VEN 400 mg cohort is now beginning. None of the pts required a second ind course and no tumor lysis was seen. The median time to ANC and platelet recovery to 1K/ul and 100K/ul in the 7 pts who received all planned VEN is 36 d (range 24-43) and 27 d (range 24-55), respectively. The table lists the specific pt characteristics, adverse events and outcome. Conclusions: We have preliminarily shown that VEN 200 mg/d beginning one d prior and until three d after 3+7 ind can be given safely in pts age 18-60; escalation to VEN 400 mg is ongoing. Flow based MRD-negative remissions have been noted after a single ind cycle. Plans are underway to compare the VEN/3+7 ind regimen derived from this trial to 3+7 in non-favorable risk newly diagnosed AML pts ages 18-60 in the context of a North American Intergroup study. Table Disclosures Stone: Novartis: Consultancy, Research Funding; Macrogenics: Consultancy; Astra-Zeneca: Consultancy; Arog: Consultancy, Research Funding; Arog: Consultancy, Research Funding; Otsuka: Consultancy; Agios: Consultancy, Research Funding; Actinium: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Other: DSMB; Daiichi-Sankyo: Consultancy; Astellas: Consultancy; Otsuka: Consultancy; Biolinerx: Consultancy; Argenix: Other: DSMB; Amgen: Membership on an entity's Board of Directors or advisory committees; Argenix: Other: DSMB; Stemline: Consultancy; Biosight: Consultancy; Roche: Consultancy; Takeda: Other: DSMB; Novartis: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Pfizer: Consultancy; Trovagene: Consultancy; Biolinerx: Consultancy; Jazz: Consultancy; Trovagene: Consultancy. DeAngelo:Incyte: Consultancy; Amgen: Consultancy; Jazz Pharmaceuticals Inc: Consultancy; Celgene: Consultancy; GlycoMimetics: Research Funding; Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Shire: Consultancy; Takeda Pharmaceuticals: Consultancy; Abbvie: Research Funding. Galinsky:Merus Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; AbbVie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; ABIM: Other: Member on specialty oncology board. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding; Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member. Konopleva:Kisoji: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding. OffLabel Disclosure: Venetoclax is being used off label in combination with standard and induction and consolidation chemotherapy in AML

Introduction: Conventional CAR-T cells express a single chain antibody variable fragment that restricts recognition to one tumor antigen and a limited set of cancers. This study employs a novel CAR fusing full-length human NKG2D with the CD3z signaling domain. In autologous transduced CM-CS1 T cells, NKG2D CAR receives endogenous costimulation via DAP10 to target multiple NKG2D-ligands that are upregulated in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. Methods: A phase 1 dose-escalation study to establish safety and feasibility of a single infusion of CM-CS1 T cells without lymphodepleting conditioning enrolled subjects with AML/MDS-RAEB or relapsed/refractory progressive multiple myeloma (MM) without standard therapy options (NCT02203825). Eligibility criteria included suitable organ function, no CNS disease, no prior allogeneic SCT or adoptive T-cell therapy, no therapy within 3 weeks prior to infusion, no immune suppression, and no uncontrolled infection. Dose-escalation spanned 4 cohorts [half-log increments from 1x106 to 3x107 CM-CS1 T cells] according to a 3+3 design. DLTs included ≥ Grade 3 non-hematologic toxicity or ≥ Grade 2 autoimmune toxicity related to CAR T cells. Initial assessment was at 28 days. At least 1 AML/MDS and 1 MM subject were mandated in each dose level. Manufacturing included PBMC stimulation with OKT3 and IL-2 followed by 2 rounds of retroviral transduction at DFCI's Cell Manipulation Core Facility. Vector copy number (VCN) and replication-competent retrovirus (RCR) testing were performed on whole blood and PBMCs, respectively, using quantitative PCR. Results: From April 2015 to July 2016, 11 subjects were infused, and 10 completed the DLT period. Eight of 11 were male, 6 had AML/MDS, and median age was 70 (range 44 to 79) (Panel A). Median WBC was 2.3 (range 0.7 to 7.2 K/uL); median ALC was 0.74 (range 0.09-2.37 K/uL). Five had cells manufactured from peripheral blood; 6 underwent apheresis. Median percentage of blasts in bone marrow for AML/MDS patients was 50% (range 4-68%). All myeloma patients had undergone ≥ 5 therapies including ≥1 autologous SCT. Four of the 6 AML/MDS patients had secondary disease, 3 had complex cytogenetics, 3 had p53 mutations, and 1 had a FLT3-ITD mutation. Dose-escalation proceeded from 1x106 to 3x107 CM-CS1 T cells. All 11 products passed release criteria, and there were no infusion reactions. Products consisted of median 97.2% CD3+ cells and 31.0% CD8+ cells, with vector-specific NKG2D expression on median 74.6% of CD3+ and 66.3% of CD8+ cells (Panel B). The first 10 subjects completed their 28 day evaluation period without DLTs. There were no cases of cytokine release syndrome, cell-related neurotoxicity, auto-immunity, or CAR T-related death. SAEs included a Grade 4 intracochlear bleed and an episode each of grade 4 neutropenia and thrombocytopenia deemed related to disease progression. Forty percent of patients experienced some Grade 3 toxicity, all related to underlying disease or a complication thereof (Panel C). At these initial cell doses, no patient to date has had objective tumor response at the 28 day evaluation mark. Nine initiated subsequent therapies; there have been 4 deaths secondary to disease or complications of subsequent therapies. However, cases of unexpected survival without further therapy and responses to subsequent treatments were noted. For example a patient with p53-mutated AML survived 4 months despite 50% blasts at infusion, and another entered PR at 6+months after cells on an IDH-1 inhibitor with Conclusion: In the first 3+ dose-escalation cohorts of patients with AML/MDS and myeloma, a single dose of CM-CS1 T cells without lymphodepletion was feasible and well-tolerated, with no DLTs. CAR T cells generally have not persisted beyond 1 week, consistent with pre-clinical models. Correlative analyses including post-infusion immunophenotyping are in process. Future studies of multiple infusions of NKG2D CAR T cells in both hematologic malignancies and solid tumors at the higher cell doses associated with efficacy in pre-clinical models are in planning. Table Table. Disclosures Murad: Celdara Medical, LLC: Employment. Reder:Celdara Medical, LLC: Employment. Sentman:Celdara Medical, LLC: Membership on an entity's Board of Directors or advisory committees, Other: Holds patents on this technology. Wade:Celdara Medical, LLC: Employment. Schmucker:Celdara Medical, LLC: Employment. Lehmann:Celyad, SA: Employment. Snykers:Celyad, SA: Employment. Allen:Celyad, SA: Employment. Stone:Celator: Consultancy; Jansen: Consultancy; Novartis: Consultancy; Merck: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Roche: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Xenetic Biosciences: Consultancy; Agios: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Pfizer: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Consultancy. Dranoff:Novartis: Employment. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees.

Background: The prognosis for patients with relapsed acute myeloid leukemia (AML) remains extremely poor. Standard reinduction regimens, such as mitoxantrone, etoposide, and ara-C (MEC), have been associated with complete remission rates in the 23 to 30% range. Lenalidomide (Len), an immunomodulatory drug, has efficacy in relapsed AML and has been shown to augment the effects of standard chemotherapies (anthracyclines and ara-C) in preclinical AML studies. These data prompted the current phase 1 study of Len in combination with MEC in pts with relapsed AML. Methods: The trial included pts (≥ 18 yrs) with relapsed or refractory AML. The primary objective was determination of the maximum tolerated dose (MTD) of Len when given in combination with MEC. Secondary objectives included safety, efficacy and time to count recovery. The study examined escalating doses (5-10 mg) of Len given daily for the first 14 days in combination with standard MEC doses of mitoxantrone (8 mg/m2/d), etoposide (100 mg/m2/d), and ara-C (1000 mg/m2/d) on days 4 through 8. Due to observations of prolonged count recovery, the Len dosing schedule was amended by reducing the duration of treatment to 10 days starting on Day 1. The dose of len was then re-escalated starting at 5 mg/d (5-10-25-50). A standard 3+3 dose-escalation design was used. Dose limiting toxicity was defined as Grade IV rash or Grade IV neuropathy during the first 28 day period, or delayed neutrophil (ANC Results: A total of 33 pts have been enrolled in the study (25 escalation phase [5 mg days 1-14, n=3; 10 mg days 1-14, n=6; 5 mg days 1-10, n=3; 10 mg days 1-10, n=3; 25 mg days 1-10, n=3; 50 mg days 1-10, n=6] and 8 expansion phase pts at 50 mg/d days 1-10). One patient was enrolled, found to be ineligible, and not treated. The median age was 62 years (range, 28-74), 45% were male and the median number of prior treatments was 2. Three dose limiting toxicities, all due to delayed count recovery past day 45, were observed in the escalation phase (n = 2/6 pts in the 10 mg/d day1-14 dose group and n = 1/6 in the in the 50 mg/d day1-10 dose group). The RP2D of Len in combination with MEC was 50 mg/d on days 1-10. Among the 14 pts treated at the RP2D, common grade 3/4 adverse events (AEs) regardless of causality were mainly hematologic including febrile neutropenia (36%). Overall 4 pts including none treated at the RP2D died during treatment. All of the on-treatment deaths, (grade 5 sepsis, n =1; infection, n=1; respiratory failure, n =2), were determined to be unrelated to the study drug. 32 pts were evaluable for response (one pt too early for assessment) with 12 pts achieving a CR and 1 pt a CRi with a total CR/CRi rate of 41% (95% CI: 24-59%). The median time to a neutrophil count of 1500/mL was 30 days and to a platelet count of 100,000/mL was 22 days. Conclusions: Len in combination with MEC re-induction chemotherapy for pts with relapsed or refractory AML was generally well tolerated and associated with an improved response rate as compared to historical controls. The safety profile was consistent with reported events in other MEC chemotherapy trials. Overall, these data suggest further exploration of high-dose Len in combination with MEC. Disclosures DeAngelo: Celgene: Consultancy; Agios: Consultancy; Incyte: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Off Label Use: lenalidomide in AML. Fathi:Takeda Pharmaceuticals International Co.: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Exelexis: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy. Steensma:Incyte: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onconova: Consultancy. Attar:Agios: Employment. Stone:Abbvie: Consultancy; Amgen: Consultancy; Agios: Consultancy; Celator: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Novartis: Research Funding; Celgene: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Roche/Genetech: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy.

B-cell receptor (BCR) signaling has been inferred as an important mechanism for disease progression in chronic lymphocytic leukemia (CLL) and other B-cell malignancies. In response to BCR activation, CLL cells secrete the chemokine CCL3, which fosters interactions between CLL cells and the leukemia microenvironment. CCL3 secretion correlates with expression of the 70-kDa ζ-associated protein (ZAP-70) and responsiveness of the CLL clone to BCR stimulation. Here, we measured CCL3 plasma levels by enzyme-linked immunosorbent assay (ELISA) in 351 CLL patients and examined CCL3 levels for associations with established prognostic markers and time from diagnosis to initial therapy. We found that CCL3 plasma concentrations were strongly associated with established prognostic markers. In a Cox proportional hazards regression model, CCL3 as well as established prognostic markers (immunoglobulin heavy chain variable-region mutation status, CD38 or ZAP-70 cytogenetics, clinical stage) were significantly associated with time to treatment. Multivariable analysis revealed that CCL3 (hazard ratio [HR] = 2.33, P < .0001), advanced clinical stage (HR = 2.75, P = .0025), poor risk cytogenetics (del 17p, HR = 2.38; del11q, HR = 2.36, P = .001), and CD38 expression (HR = 1.43, P = .023) were independent prognostic markers. Collectively, CCL3 is a novel, robust, and independent prognostic marker in CLL that can easily and reliably be measured by ELISA. CCL3 therefore should become useful for risk assessment in patients with CLL.

Introduction: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765) has proved to be an effective targeted therapy in patients with chronic lymphocytic leukemia (CLL). A multicenter phase Ib/II study of ibrutinib in 85 patients with relapsed CLL or small lymphocytic lymphoma (SLL) showed the overall response rate of 71% with an additional 15 to 20% of patients having a partial response with lymphocytosis. A phase II trial of ibrutinib in combination with rituximab in 40 high-risk CLL patients conducted at MD Anderson Cancer Center demonstrated an overall response rate of 95%. However efficacy of ibrutinib in combination with rituximab vs. ibrutinib alone has not been directly compared. A randomized study of ibrutinib vs. ibrutinib+rituximab in patients with relapsed CLL was designed to compare long-term progression free survival rate in two groups (NCT02007044). Here we test a hypothesis that addition of rituximab may abrogate ibrutinib-induced lymphocytosis and accelerate response of other biomarkers, including plasma levels of chemokines CCL3 (MIP-1a) and CCL4 (MIP-1b), which are known to reflect the activation status of CLL cells. Methods: Patients were randomized to ibrutinib alone (arm A) or ibrutinib plus rituximab arm (arm B). Patients on arm A were treated with ibrutinib 420 mg orally once daily continuously throughout the study. Patients randomized to arm B received ibrutinib starting at day 1 or day 2. Rituximab (375 mg/m2) was administered intravenously on days 1, 8, 15, and 22 (cycle 1), then monthly during cycles 2-6. Study inclusion required previously treated CLL/SLL or treated or untreated high-risk disease (del17p or TP53 mutation). Serial blood samples were drawn at pre-treatment, then weekly during the first month, and monthly until 5/6 months. Plasma levels of CCL3 and CCL4 were measured at pre-treatment, 1 week, 1 month, 3 months and 5 months of follow-up by ELISA. A linear mixed effects model with randomly varying intercepts and slopes was constructed for each absolute lymphocyte count (ALC) measured repeatedly over time using SAS PROC MIXED. Models included an unstructured covariance and the Kenward-Rogers degrees of freedom adjustment to take into account unequally spaced unbalanced data. Models over at least 4 months of measurements included both linear and quadratic components. Results: Accrual began in December 2013; currently 93 of 208 patients are enrolled. 45 patients (48%) were randomized to arm A and 48 (52%) to arm B. Median ALC at pre-treatment was similar on both arms (26.28 and 25.14 K/µl). A mixed model with quadratic trend was used to assess the dynamic changes in ALC during the first month of treatment. The model confirmed a curvature pattern for arm A, consistent with lymphocytosis: ALC rose immediately after initiation of treatment and then began to stabilize. In arm B, however, no statistically significant changes in ALC were observed during this initial period. To appreciate the difference between two treatment arms over a longer time period, changes in ALC were analyzed in the 52 patients who had 4 or more months of follow-up. A mixed model with a quadratic trend demonstrated ALC downward trend for both arms, with a more rapid decrease in arm B (p=0.03 for the linear component; p=0.004 for the quadratic). The curvature in ALC reflecting initial lymphocytosis was clear in arm A, but not arm B. Descriptive analysis suggested that arm B had higher percentage of patients with normal ( Concentration of CCL3 and CCL4 was significantly reduced after one week of treatment on both arms and remained low (Figure 1). Formal statistical comparison of CCL3 and CCL4 patterns is in process. Conclusion: In this randomized evaluation of ibrutinib vs. ibrutinib+rituximab an initial analysis suggests that addition of rituximab to ibrutinib abrogates the transient lymphocytosis and is associated with faster clearing of the bloodstream of leukemia cells in patients with relapsed CLL. Significant reduction of CCL3 and CCL4 levels was observed on both treatment arms. Figure 1 Figure 1. Disclosures Kipps: Pharmacyclics: Research Funding. O'Brien:Pharmacyclics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Burger:Pharmacyclics: Consultancy, Honoraria, Research Funding.

Background: CCL3 and CCL4, previously called macrophage inflammatory protein-1 alpha (MIP-1α) and MIP-1β, are chemokines of the CC subfamily, which are secreted by CLL cells in response to B cell receptor (BCR) stimulation and by co-culture with nurse-like cells (NLC), presumably to attract T lymphocytes and monocytes that can deliver pro-survival signals. We previously reported, that CLL patients have elevated plasma levels of CCL3 and CCL4, which rapidly normalize after treatment with kinase inhibitors that target BCR signaling, such as the BTK inhibitor ibrutinib and the PI3K inhibitor idelalisib (Blood 119:1182-9, 2012)(Blood 118:3603-12, 2011). Moreover, in untreated CLL patients, high CCL3 levels are an independent prognostic marker for short time to disease progression (Blood 117:1662-9, 2011), emphasizing the clinical relevance of these chemokines in CLL. Aim: To better understand the dynamic changes, we analyzed plasma levels of CCL3 and CCL4 in a large cohort of serial samples from untreated CLL patients. Additionally, we analyzed separately from the entire cohort, CCL3 and CCL4 changes overtime in patients who subsequently received therapy and patients who were still on observation at time of analysis. Methods: CCL3 and CCL4 plasma levels were measured by ELISA in serial samples collected over time in untreated CLL patients. A total of 193 individual patients with 603 plasma samples were included in the analysis (entire cohort), of the 193 patients, 81 of them required treatment at a later time, although all the samples analyzed were collected prior any treatment, 112 patients did not need any intervention over the period of sample collection and were managed with observation. The duration between first and subsequent samples ranged between 5 months to 12 years. The duration between the date of the last sample and the date of the initiation of treatment ranged between less than a month to 88 months with a median of 6 months. Mixed linear models with repeated measure were used to determine whether there was a difference in mean levels of CCL3 and CCL4 over time. Results: The median and range of CCL3 and CCL4 levels for the initial sample over all 193 patients were 11.8 pg/mL (2.5 – 1977.1 pg/mL) and 88.8 pg/mL (6.9 – 2601.3 pg/mL) respectively. There were significant associations of time between first and subsequent serial samples (p Conclusions: These studies demonstrated that there is a slight but statistically significant increase in the levels of the chemokines CCL3 and CCL4 over time in CLL patients, regardless of whether the patients required therapy at a later time. We conclude that CCL3 and CCL4 plasma levels presumably reflect the dynamic changes of CLL pathogenesis over time, strengthening the relevance of CCL3 and CCL4 chemokine production in CLL. Disclosures No relevant conflicts of interest to declare.

Constitutive activation of the NF-κB pathway is a critical feature of hematologic malignancies and is associated with increased lymphocyte proliferation and survival. Various mechanisms leading to altered NF-κB activation have been described in lymphomas, including activating mutations in and upregulation of NF-κB pathway genes. We have previously reported whole-exome sequencing results from a cohort of 160 CLL patients showing mutations in several NF-κB pathway genes (as defined in the Ingenuity Pathway Analysis database), including MYD88 (n=12), RIPK1 (n=4), NRAS (n=4), KRAS (n=3), CARD11 (n=1), IRAK4 (n=1), PIK3CA (n=1) and TRAF3 (n=1). Here, we describe functional approaches to evaluate the biological significance of NF-κB pathway mutations. Using the CellTiter-Glo assay in serum-free conditions, we assessed the relationship between NF-κB pathway mutation status and survival of CLL cells in vitro. We found that CLL cells with no NF-κB mutations exhibited spontaneous apoptosis in vitro, with 3.3 - 39.5% viable cells remaining after 48 hours in culture (n=4 patient samples; n=5 replicates per patient). Similarly, cells harboring the MYD88 L265P mutation yielded 0.6 - 23.1% viable cells after 48 hours (n=5 patient samples; n=5 replicates per patient). In contrast, cells harboring RIPK1 Q375*, RIPK1 K559R, KRAS Q61H, CARD11 E756K, IRAK4 K400E, and PIK3CA I143V displayed apoptotic resistance, with 48.6 - 132.5% viable cells remaining after 48 hours (1 patient sample per mutation; n=5 replicates per patient). Similar findings were observed in the context of B-cell receptor activation via IgM stimulation, with IgM stimulation generally enhancing CLL cell viability (mean = 24% increase after 48 hours). In the case of RIPK1 Q375*, however, no further increase in viability was observed, suggesting that this truncating mutation may obviate the need for external survival signals. To identify NF-κB mutations that might associate with susceptibility to different therapies, we examined the relationship between NF-κB pathway mutation status and sensitivity to the NF-κB inhibitor, SN50. Treatment with SN50 for 48 hours resulted in loss of viability in cells that were negative for NF-κB pathway mutations (43.3 - 98.9% decrease in viability with 5 μM SN50; n=4 patient samples; n=5 replicates per patient). Similarly, cells harboring the MYD88 L265P mutation responded to SN50 treatment (43.1 - 97.0% decrease in viability; n=5 patient samples; n=5 replicates per patient). In contrast, cells harboring RIPK1 Q375*, RIPK1 K559R, CARD11 E756K, and PIK3CA I143V were more resistant to SN50 treatment (18.6% increase - 38.9% decrease in viability; 1 patient sample per mutation; n=5 replicates per patient). These results suggest that specific NF-κB pathway mutations confer resistance to NF-κB inhibition. The above results prompted the question of whether NF-κB pathway mutations may also confer resistance to the BTK inhibitor, ibrutinib, which has previously been shown to block NF-κB pathway activation by inhibiting the phosphorylation of p65 and preventing its nuclear translocation. To address this question, we treated cells with 2.5 μM ibrutinib for 48 hours. Ibrutinib treatment led to a loss in viability in cells that had no NF-κB pathway mutations (26.3 - 82.9% decrease in viability; n=4 patient samples; n=5 replicates per patient). Cells harboring MYD88 L265P mutations also appeared to be susceptible to ibrutinib (25.8 - 82.9% decrease in viability; n=5 patient samples; n=5 replicates per patient). Notably, cells harboring the RIPK1 Q375* and KRAS Q61H mutations appeared to be more resistant to treatment (7.0% decrease and 2.0% increase in viability, respectively; 1 patient sample per mutation; n=5 replicates per patient). Furthermore, RIPK1 Q375* and KRAS Q61H cells remained resistant to ibrutinib in the context of BCR activation. Though our study utilized a limited number of patient samples representing a variety of mutations, the results are suggestive that specific NF-κB pathway mutations are functional and may influence intrinsic CLL cell survival, responsiveness to IgM stimulation, and sensitivity to drug treatment. Identification of specific mutations that confer resistance to ibrutinib is of particular clinical interest for predicting response and understanding drug resistance. Disclosures: Brown: Novartis: Consultancy; Vertex: Consultancy; Sanofi Aventis: Consultancy; Onyx: Consultancy; Emergent: Consultancy; Celgene: Consultancy, Research Funding; Genentech: Consultancy; Pharmacyclics: Consultancy; Genzyme: Research Funding.

Recent work in CLL and other lymphoproliferative disorders has established the importance of somatic mutations in the pathogenesis and prognosis of disease. MYD88 L265P mutations occur in approximately 30% of ABC diffuse large B cell lymphomas (ABC DLBCL) and 90% of Waldenstrom’s macroglobulinemia (WM) cases and have been shown to be an activating mutation in both cancers. MYD88 is an important adaptor protein for IL-1 and many Toll-like receptors, which signal via dimerization followed by formation of a complex of MYD88 with IRAK4 and IRAK1, leading to activation of IRAK4 kinase activity, IRAK1 phosphorylation and downstream activation of NFκB. Previous work in CLL has demonstrated that MYD88 L265P mutations occur in a smaller subset of patients (2.9-6.25%) and although MYD88 mutation is thought to be a CLL driver mutation, its role in CLL is not clearly understood. We previously reported whole exome sequencing results from a cohort of 160 CLL patients, 10 of whom have MYD88 L265P mutation (6.25%). Eight of ten patients with MYD88 L265P mutation had mutated IGHV, consistent with prior reports of this association. However, patients with MYD88 L265P mutation did not have a younger age at diagnosis, possibly due to the overall younger median age of our patient cohort (54, range 34-77). RNA gene expression profiling of 146 CLL and 20 normal B cell samples revealed that normalized median expression of IRAK1, IRAK4 and NFKB1 appear lower in CLL samples versus normal B cells (IRAK1 p = 0.0001, IRAK4 p = 0.01, NFKB1 p = 0.0005). However, median expression levels of MYD88 appear higher in CLL samples versus normal B cells (p = 0.006). Although MYD88 L265P mutation has been shown to activate BTK in WM, mean BTK protein levels measured by flow cytometry were similar between CLL patients with and without MYD88 mutation (mean % cells positive for BTK, 98.3% in MYD88 L265P mutants vs. 98.16% in wild-type [WT], 3 patients/group), as were levels of phosphorylated BTK (mean % cells positive, 13.28% in MYD88 L265P vs. 14.35% in WT). Chronic BTK-mediated B cell receptor signaling is a hallmark of CLL and studies with the BTK inhibitor ibrutinib (PCI-32765) have demonstrated encouraging therapeutic results in vitro and in vivo. ND-2110 and ND-2158 are potent, highly selective IRAK4 kinase inhibitors which have single agent activity in vitro in ABC DLBCL cell lines as well as synergistic effects with BTK inhibitors. Given these findings, ND-2110 and ND-2158 were ideal candidates to test in CLL. As such, CLL samples with and without MYD88 L265P mutation (3 per group) were cultured in vitro in the presence of 5μM ND-2110 or ND-2158 as single agents or in combination with 2.5μM ibrutinib for 72 hours. A second, higher dose combination was also tested, using 25μM ND-2110 or ND-2158 and 3.75μM ibrutinib. Viability was assessed using Cell TiterGlo and normalized to DMSO treated cells. A general linear model was used to assess the association of % viability with MYD88 L265P mutation, IgM stimulation and combinations of IRAK4 inhibitor compounds and ibrutinib. MYD88 L265P was associated with higher viability, hence less cell killing (p = 0.002 5μM ND-2110 & 2.5μM ibrutinib; p < 0.0001 5μM ND-2158 & 2.5μM ibrutinib; p = 0.005 25μM ND-2158 & 3.75μM ibrutinib). Cells treated with 25μM ND-2158 + 3.75μM ibrutinib had significantly lower viability compared to cells treated with 25μM ND-2158 alone (p < 0.0001). IgM stimulation also resulted in lower viability compared to no stimulation, suggesting sensitization (p = 0.002 25μM ND-2100 + 3.75μM ibrutinib; p < 0.0001 all other combinations). Ibrutinib appeared to have less single agent activity in MYD88 L265P mutants, while the activity of single agent ND-2110 and ND-2158 was more equalized between groups. These preliminary results are currently being confirmed in additional patients. To further characterize the effects of these drugs, we have established a model system in which CLL cells are co-cultured with NIH3T3 cells expressing CD40L, with and without CpG stimulation. Preliminary results show that in one WT MYD88 CLL patient, these conditions resulted in significant induction of Ki67 expression, which was reduced by combination of either ND-2110 or 2158 with ibrutinib. These results suggest that IRAK4 inhibitors in combination with ibrutinib have interesting activity in CLL, which may differ based on MYD88 L265P mutation; experiments are in progress in additional patients to further characterize this effect. Disclosures: Chaudhary: NIMBUS Discovery: Employment. Romero:NIMBUS Discovery: Consultancy, Equity Ownership. Robinson:NIMBUS Discovery: Consultancy. Westlin:NIMBUS Discovery: Employment. Brown:Pharmacyclics: Consultancy; Genentech: Consultancy; Celgene: Consultancy, Research Funding; Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding.

Survival, proliferation, and resistance to chemotherapy in CLL cells have been shown to be consistently associated with the activity of the B-cell receptor (BCR) and the associated downstream pathways activated by the BCR. Key molecules in this pathway are LYN and SYK (Spleen tyrosine kinase), as well as PI3K, BTK (Bruton’s tyrosine kinase), and others. Dasatinib, given at standard doses, allows for serum levels well above 11 nM, the IC50 for suppression of LYN kinase. We have previously shown that dasatinib used as a single agent in patients with relapsed CLL results in lymph node responses in 60% of patients and partial responses in 20% of patients as defined by NCI-WG criteria. In the current study, patients with relapsed CLL were treated with a regimen combining dasatinib at 140 mg/day, days 1-14, with fludarabine (F) 25 mg/m2/day, days 1-3, and rituximab (R) 375 mg/m2 per cycle repeated every 28 days, while effective up to 6 cycles. Patients were followed closely for response with CT scans every 2 months initially. Among the first 10 patients treated, all had responses according to IWCLL criteria as follows: The median time to progression was 21 months. In the first week multiple blood samples were taken for analysis of target inhibition and subsequent apoptosis. The schedule of administered agents was altered in the first week to determine which components were associated with which downstream effects. Hence, dasatinib was given on Day 1, no treatment was administered on Day 2, F and R without dasatinib on Day 3, dasatinib with FR on Day 4, and dasatinib with F on Day 5. Initial in vitro studies revealed inhibition of phosphorylation of Lyn at 6 hours after patients were given dasatinib on Days 1 and 4, with recovery by 24 hours. Day 3 treatment with FR but without dasatinib showed no such inhibition at 6 hours. Assessment of global tyrosine phosphorylation in CLL cells showed this same pattern, including that of Syk phosphorylation, specifically. Flow cytometry for annexin-V demonstrated that apoptosis was greatest on Day 4 after 6 hours of exposure to all 3 drugs. We conclude the following: 1) the combination of dasatinib with FR, as seen in the first 10 patients of this study, was associated with excellent responses in blood and lymph nodes as assessed by physical exam, 2) the combination was well tolerated with mainly hematologic toxicity, 3) the inhibition of phosphorylation of Lyn and Syk was associated with apoptosis and clinical response. This combination may have therapeutic promise in advanced CLL and is worthy of further investigation. Disclosures: Off Label Use: Dasatinib use in CLL is off-label. This trial shows that dasatinib may be beneficial in the treatment of CLL. Brown:Pharmacyclics: Consultancy; Genentech: Consultancy; Celgene: Consultancy, Research Funding; Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding. Fathi:Seattle Genetics, Inc.: Advisory/Scientific board membership Other, Research Funding; Millennium: Research Funding; Agios: Membership on an entity’s Board of Directors or advisory committees; Teva: Membership on an entity’s Board of Directors or advisory committees.

Abstract 2875 Genome wide association studies (GWAS) in chronic lymphocytic leukemia (CLL) have identified thirteen single nucleotide polymorphisms (SNPs) that are associated with the risk of developing CLL but do not affect the coding regions of genes. The functional targets of these SNPs remain largely unknown although they are thought to potentially serve as regulatory elements for nearby genes. We have previously published the results of a high resolution integrated genomic analysis of 161 CLLs with matched normal DNAs using Affymetrix 6.0 SNP arrays and Affymetrix U133 Plus 2.0 arrays run on the CLL lymphocytes. In this analysis, we sought to exploit this dataset to investigate whether SNP genotype at loci implicated in CLL risk by GWAS was associated with altered expression of genes in the CLL lymphocyte expression arrays. We therefore investigated 19 SNPs previously described in GWAS studies, either the SNP itself if present on the Affymetrix 6.0 SNP array, or one or more proxy SNPs for those not present on the array, chosen based on their high linkage disequilibrium (r2 > 0.7, usually > 0.9) with the GWAS SNP. Regions studied included 2q13 (1 SNP), 2q37.1 (1 SNP), 2q37.3 (1 SNP), 6p21.3 (1 SNP), 6p25.3 (2 SNPs), 8q24.2 (6 proxy SNPs), 11q24.1 (1 SNP), 15q21.3 (1 SNP), 15q23 (1 proxy SNP), 15q25.2 (1 SNP), 18q21.1 (1 proxy SNP), and 19q13.32 (2 proxy SNPs). We hypothesized that the genes most likely to be regulated by these loci would be located nearby, and therefore explored associations between SNP genotypes and the expression of genes located within 2 Mb of the relevant SNP using the Kruskal-Wallis test. The number of genes evaluated ranged from 11–22 depending on the locus. The analysis was performed independently for SNP genotypes derived from the tumor / lymphocyte samples (n=143) and from the normal / saliva samples (n=70–80). Discordant genotypes between tumor and normal samples were manually reviewed for reconciliation or excluded in the case of poor quality, indeterminate genotype or altered genomic copy number at the locus. Using the SNP genotypes from the tumor samples, we identified 13 genes with expression significantly associated with a risk SNP (using p value < 0.05). Using the SNP genotypes from the normal samples, we identified 15 genes using the same criteria. In both the tumor and normal analyses, eight SNPs were associated with a total of seven genes. The most significant associations were found between the risk allele of rs674313 on 6p21 and higher expression of HLA-DQA1 (p Disclosures: No relevant conflicts of interest to declare.

Abstract 560 Nuclear factor kappa B (NF-κB) encompasses a family of transcription factors involved in oncogenic processes including cellular proliferation and apoptotic inhibition. Constitutive activation of NF-κB has been observed in hematologic malignancies and is thought to confer resistance to chemotherapeutic agents. Here, we examine the role of the NF-κB pathway in chronic lymphocytic leukemia (CLL). Whole-exome sequencing was performed using tumor and matched germline DNA from 167 CLL patients. We identified 51 patients (30%) carrying 53 non-silent somatic variants in genes of the canonical NF-κB pathway, which consists of 272 genes as defined by the Ingenuity Pathway Analysis tool. Of the 99 patients whose germline sequences have been analyzed to date, 27 patients (27%) carry 34 non-silent germline variants in NF-κB pathway genes. A total of 67 patients (40%) have at least one non-silent somatic or germline variant. Variants in the NFKB1 gene, itself, were also observed: a somatic variant, H66R, found in two patients, and two germline variants, Y89F and R849W, each found in one patient. To evaluate the functional consequences of the NFKB1 variants, we performed site-directed mutagenesis to generate full-length NFKB1 cDNAs encoding these variants. We subsequently measured transcriptional activity of wild-type and mutant NFKB1 via luciferase assays in HEK293T cells using reporter cassettes containing the NFKB1 response element. Transcriptional activity of the three NFKB1 variants was found to be at least 2-fold higher than that of wild-type NFKB1 (p To test the hypothesis that deregulation of the NF-κB pathway is a key mechanism in CLL, we compared gene expression profiles of NF-κB pathway genes between CLL patient samples (n=146) and normal B cells (n=16) and found overall upregulation of the NF-κB pathway in CLL (Kolmogorov-Smirnov test, p=2.2e-16). K-means clustering and principal component analysis (PCA) further revealed that CLL patients can be divided into two subgroups exhibiting differential magnitude of NF-κB pathway upregulation. Studies in progress aim to identify the clinical significance of these subgroups. Finally, we assessed the effect of inhibiting the NF-κB pathway using the cell permeant NF-κB inhibitor, SN50. We performed Annexin V/PI staining 24 hours post-treatment in CLL cells with (n=9) or without (n=3) NF-κB pathway variants. SN50 increased cell death 1.8-fold in all cells tested (p In conclusion, our findings demonstrate that a high proportion of CLL patients harbor somatic and germline variants in NF-κB pathway genes, some of which appear to be functional. Furthermore, the NF-κB pathway is upregulated in CLL and pharmacological inhibition of the pathway leads to increased cancer cell death. Functional characterization of NF-κB pathway variants offers mechanistic insight into the disease, providing novel targets for therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3885 Lenalidomide has promising clinical activity in patients with chronic lymphocytic leukemia (CLL). Unlike other anti-leukemia drugs, lenalidomide is not cytotoxic for CLL cells in vitro. Similar to CD154, lenalidomide can enhance CLL-cell expression of immune co-stimulatory molecules, formation of immunologic synapse, activation of NK-cells, and generation of anti-tumor immunity. Furthermore, lenalidomide repeatedly can enhance expression of CD154, which we had observed was functionally deficient in patients with CLL. However the exact mechanism of action of lenalidomide is still under investigation. Herein, we studied the gene expression profile and microRNA (miR) of CLL cells collected from 20 patients before and at day 8 and day 15 of treatment with 2.5–5 mg of lenalidomide in the CRC014 trial. We observed significant changes in expression level of 54 genes at day 8 versus pre-treatment samples. We identified significant changes in expression level of 189 genes at day 15 versus pre-treatment samples. This included 44 of the 54 (81%) genes noted at day 8. Forty genes were expressed at significantly higher levels at day 8 and day 15 of lenalidomide treatment. We noted that 7 (17%) of these genes were related to Ras pathway and its downstream signaling pathways (i.e. NF-KappaB pathway): Ras association (RalGDS/AF-6) domain family member 4 (RASSF4), a member of RAS oncogene family (RAB13), Ras protein-specific guanine nucleotide releasing factor 1 (RASGRF1), GTPase IMAP family member 6 (GIMAP6), GTP-binding protein ras homolog gene family member S (RND1), kinase suppressor of Ras 2 (KSR2) and toll-like receptor adaptor molecule 2 (TICAM2). Ras signaling affects many cellular functions, which includes cell proliferation, apoptosis, migration, fate specification, and differentiation. In the resting cells, Ras is tightly bound to GDP (Guanosine Diphosphate), which is exchanged for GTP (Guanosine Triphosphate) upon binding to activated cell membrane receptors. In the GTP-bound form, Ras interacts with a broad range of effector proteins to induce a diverse array of biological consequences. Although typically associated with enhanced growth and transformation, activated Ras also may induce growth antagonistic effects such as senescence or apoptosis. Some of the growth-inhibitory properties of Ras are mediated via the RASSF family of Ras effector/tumor suppressors. RASSF4 is the fifth member of this family and it binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibiting the basic properties of a Ras effector. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines. Although broadly expressed in normal tissue, RASSF4 is frequently down-regulated by promoter methylation in human tumor cells and primary tumors. However changes in miR expression also may affect the level of gene expression. Therefore we analyzed miRs expression by microarray in pre treatment, day 8, and day 15 CLL samples. We observed significant changes in expression levels of 33 miRs between day 8 and pre treatment samples. We identified significant changes in expression levels of 11 miRs between day 15 and pre treatment samples. Of the 33 miRs differentially expressed at day 8, only 5 were up-regulated whereas the remaining 28 were down-regulated. Interestingly, among these 28 down-regulated miRs, 5 miRs (miR-103, miR-16, miR-30a, miR-30b and miR-342-3p) target RASSF4. Noteworthy, miR-342-3p was one of the 3 miRs (miR-26a, miR-138 and miR-342-3p) down-regulated both at day 8 and at day 15, suggesting that the down-regulation of such miR has a key role in the overexpression of RASSF4 leading to Ras-dependent apoptosis. Further studies are ongoing to elucidate lenalidomide action on CLL cells via RASSF4 overexpression. This study demonstrates that treatment with lenalidomide can induce down-regulation of miRs associated with changes in gene expression by CLL cells, leading to over-expression of RASSF4 and other Ras or GTPase related proteins that can induce growth antagonistic effects and account in part for the activity of this drug in CLL. Disclosures: James: Celgene: Research Funding. Neuberg:Celgene: Research Funding. Corral:Celgene: Employment. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Abstract 291 Lenalidomide (L) is an immunomodulatory agent with therapeutic activity in CLL; rituximab (R), a CD20 mAb, has limited activity as monotherapy in CLL. In preclinical models, L resulted in NK cell expansion and synergized with R. Therefore, a phase II, 2-stage study was designed to evaluate this combination. Treatment-naïve patients (pts) were eligible if they had an indication for therapy, normal kidney function, and no history of deep vein thrombosis or pulmonary embolic events. Pts started L at 2.5 mg per day (D) and could escalate to 5 mg/D on D8 and again on C3D1 to a maximum of 10 mg/D if tolerated. Pts received L 21 of 35 D for cycle (C) 1, then for 21/28 D for C2-7 (7 cycles total). R was started at the end of C1 at 50 mg/m2 D29, 325 mg/m2 D31, 375 mg/m2 D33, C1, then 375 mg/m2 weekly × 4 for C2 and on D1 for C3-7. Pts received allopurinol 300 mg daily, and following a protocol amendment, aspirin at 81 mg daily. The study has closed to accrual with 69 pts enrolled at four CRC clinical sites. 40 pts enrolled into arm A (age under 65). 29 in arm B (age 65 or older). 57 now have final response data. The median age on arm A was 57 years (range 45–64) and arm B 70 years (range 65–80). Advanced Rai stage (III-IV) was present at baseline more frequently in arm B pts (48%) compared to arm A pts (23%) (p= 0.04). 73% of pts on arm A had an excellent performance status (ECOG 0/5) compared to only 54% of pts on arm B. Arm A pts frequently had aggressive leukemia features including unmutated IgVH genes and/or elevated CLL expression of ZAP-70 in 70%, these features were present in 52% of those on arm B. 4 pts on arm A and 2 on arm B had del(17p) by FISH whereas 5 pts in arm A and 4 in arm B had del(11q). A significantly lower proportion of pts on arm B (16/27) were able to complete the 7 cycles of therapy compared to (35/39) of eligible pts on arm A (p=0.013) mainly due to toxicity. Similarly, older patients were limited in dose escalation or maintenance of the maximum L dose (10 mg). 27/39 pts on arm A achieved a median L dose of 10 mg while only 13/27 pts on arm B tolerated the 10 mg L dose. The most frequent and severe adverse events (AEs) are summarized in Table 1. Infusion and tumor flare reactions (TFR) were observed more frequently during cycle 1 than in subsequent cycles. Neutropenia was frequently grade (G) 3/4 in severity. Severe (G3/4) AEs were reported in 72% of arm A pts and 82% of pts on arm B (p =.55), with non-heme G3/4 toxicities reported in 37% of arm A and 52% of arm B (p =.31).Table 1Frequent and Severe AEsArm AArm BGradeI/IIIII/IVI/IIIII/IVTFR32–161Neutropenia1119115Anemia153141Thrombocytopenia211131Fatigue25–142AST/ALT elevation183113Hypophosphatemia19271Hypocalcemia21–15–Infusion Reaction20–53Respiratory Infection17–5–Pneumonia–123Rash142121Neutropenic Fever–2–2PE/DVT–––2 Responses are summarized in Table 2. The overall response rate (ORR) to therapy for arm A was 94%, 90% CI (83%, 99%) with 20% (10%, 34%) achieving a complete remission (CR) and 17% a nodular PR (nPR). The ORR for arm B was 77% with 90% CI (58%, 91%) with 9% (2%, 26%) achieving a CR. Arm A patients had median follow-up of 17 months (from D 1 of treatment) with an estimated median progression free survival of 19 months. Arm B had a median follow-up of 7 months, with an estimated 85% remaining progression free at 7 months.Table 2ResponsesArm A (Age < 65) N = 35Arm B (Age >= 65) N = 22NCR N (%)nPR N (%)PR N (%)ORR N (%)NCR N (%)PR N (%)ORR N (%)All patients357 (20)6 (17)20 (57)33 (94)222 (9)15 (68)17 (77)IgVH unmutated224 (18)2 (9)15 (68)21 (96)131 (8)10 (77)11 (85)IgVH mutated133 (23)4 (31)5 (38)12 (92)91 (11)5 (56)6 (67)Median L dose 10 mg247 (29)5 (21)12 (50)24 (100)82 (25)5 (63)7 (88)Rai stage III/IV92 (22)1 (11)5 (56)8 (89)111 (9)6 (55)7 (64)17p del3002 (67)2 (67)100011q del31 (33)02 (67)3 (100)403 (75)3 (75)TFR present285 (18)5 (18)16 (57)26 (93)14011 (79)11 (79) A defined course of seven cycles of L and R administered for the initial treatment of CLL was associated with a high response rate (ORR 88%) with 16% CRs and 11% nPRs. Older patients were more likely to have advanced Rai stage at baseline and were less likely to escalate or maintain the maximum L dose and/or complete the 7 cycles of therapy, factors that may have contributed to a lower CR, and ORR rate in arm B. An additional 11 patients will have final response assessments by October 2011. Disclosures: James: Celgene: Honoraria, Research Funding. Off Label Use: Lenalidomide for Chronic lymphoyctic leukemia. Brown:Calistoga: Consultancy; Pharmacyclics: Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Research Funding. Rai:Celgene: Membership on an entity's Board of Directors or advisory committees. Neuberg:Celgene: Research Funding. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Abstract 465 CLL is among the most heritable of all cancers. To understand the genetic basis of this heritability, we have undertaken a comprehensive genomic analysis of familial CLLs including copy number analysis, gene expression profiling (GEP) and whole exome sequencing (WES). First, we examined whether familial and sporadic cases differ in the spectrum of acquired somatic mutations by WES of tumor and germline DNA of 36 familial CLLs (from 31 affected families). Compared to 55 sporadic CLLs, we observed that the somatic mutation rate in the familial CLLs was similar (mean 0.89 mutations/Mb (range 0.29–3.06) for the sporadics vs mean 0.97/Mb (range 0.11–3.78) for the familials, p=0.40). We also examined the spectrum of somatic mutations by testing for enrichment of 9 recently identified putative tumor drivers in our large CLL sequencing study (reported elsewhere in this meeting). We observed a similar distribution of these recurrent CLL mutations among the 36 familial CLLs as the 55 sporadic CLLs. These results were further confirmed by genotyping of the CLL driver mutations in an additional 32 familial and 67 sporadic CLLs. Collectively, these studies suggest that while the predisposing germline events may differ between familial and sporadic CLL, the spectrum of mutations and pattern of mutagenesis appear similar in the established CLL tumors. We therefore proceeded to examine the genetic characterization of germline DNA to identify predisposing loci, which we hypothesized might be enriched in a familial disease context. We first examined germline copy number variations (CNVs), which have not been previously characterized in this disease. We used high resolution Affymetrix 6.0 SNP arrays to study both tumor and germline DNA of 58 individuals representing 44 different families with CLL and lymphoproliferative disorders (LPDs). We identified two families (A and B) with autosomal dominant inheritance of CLL who carried distinct germline CNVs that affect genes previously implicated in CLL. Members of Family A carried a 525 kb germline deletion targeting DLEU7 at 13q14, but not affecting DLEU2, miR-15a, or miR-16–1. Importantly, by examining the tumor genome from these family members, we observed a uniform loss of the second allele of DLEU7 in 2/2 available CLLs from this family, suggesting an acquired “second hit” of a tumor suppressor gene. These findings underline the complexity of the most common somatically acquired copy number aberration (CNA) in CLL, 13q14 deletion, by demonstrating the role of additional regions other than the heavily investigated miRNA cluster. Members of Family B carried a 720 kb germline gain of 6p25 affecting the IRF4 gene, previously implicated in CLL through the identification of a GWAS risk allele located in the 3' UTR of IRF4, as well as the recent description of a recurrent somatic mutation affecting 1.5% of CLL cases. In Family B, the coding regions of the four genes located in this 6p gain, namely IRF4, DUSP22, EXOC2 and HUS1B, were sequenced, and no somatic mutations or novel SNPs were identified. However, the 6p gain in Family B represents an allele-specific enrichment of the haplotype carrying the GWAS risk SNP and, as previously described for that allele, results in lower expression of IRF4 in the two CLLs tested in this family. GEP further identified a signature associated with 6p gain that preserved low expression of IRF4 and showed high expression of KLF6. These results demonstrate that germline CNVs may facilitate the “path to cancer” by providing either an allelic deletion of a tumor suppressor or an amplification of a risk allele. As most familial CLL cases have not been accounted for by known SNPs or germline CNVs, we have initiated an in depth analysis of the WES germline results from familial cases compared to both sporadic CLL patients and normal individuals. Candidate variants have been filtered to exclude all SNPs described in the 1000 Genomes project and to focus on highly conserved sites. Thus far we have found that rare germline variants in patients with familial CLL contain a rich source of loci with relevance to B cell biology. Studies in progress are focused on further analysis of informative families and functional analyses of candidate variants. These comprehensive genomic analyses are expected to identify multiple cooperating genetic mechanisms that contribute to CLL pathogenesis, including CNVs and somatic and germline mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract 1438 The hierarchical cytogenetic classification for chronic lymphocytic leukemia (CLL) provides a valuable prognostic framework validated in several studies; however, the relationship between these cytogenetic abnormalities and IGHV and ZAP-70 has not been as well studied. Less is also known about the prognostic significance of different 14q32 rearrangements and combined cytogenetic abnormalities. We retrospectively examined cytogenetic abnormalities in 576 CLL patients seen at the Dana-Farber Cancer Institute between 2005 and the present. Interphase FISH analysis was performed using commercial probe sets (Abbott Molecular, Des Plaines, IL, USA) including P53/ATM, 12cen/13q14.1, and CCND1-IGH to exclude mantle cell lymphoma and identify chromosome 14q32 rearrangements, which were further evaluated with a t(14;18) probe. t(14;18) was evaluated separately from other 14q32 rearrangements. Fisher's exact test was used to assess the association of ZAP-70 and IGHV mutation status with cytogenetics. Median time to first treatment (TTFT) was summarized using Kaplan Meier estimates. In univariate analysis, the log rank test was used to assess associations of TTFT and cytogenetics, and in multivariable analysis, a Cox regression model was used. P-values were not adjusted for multiplicity of testing. The median age at diagnosis was 55 (range 25–86). At time of analysis, 43% (n=247) of patients had been treated, and 78% (n=452) of patients were still alive. Of 432 patients tested, 57.4% (n=248) had mutated IGHV, and of 420 patients tested, 42% (n=243) were ZAP-70 positive. Cytogenetic abnormalities of the 576 patients are summarized in table 1. Patients with 2 abnormalities were classified as the poorer risk abnormality by the Dohner hierarchy. The del(13q) group was significantly associated with negative ZAP-70 status (p = 0.003), whereas del(11q) was associated with positive ZAP-70 (p=0.02). A significant association was observed between unmutated IGHV and del(17p), del(11q), and trisomy 12 (p < 0.001 for each), and with complex cytogenetics (p = 0.03), whereas del(13q) had a significant association with mutated IGHV (p < 0.0001). In univariate analysis, a shorter median TTFT was associated with del(17p) vs. other (44 vs. 77 months, p = 0.0006), del(11q) vs. other (22 vs. 77 months, p < 0.0001), and 14q32 rearrangement vs. other (44 vs. 76 months, p=0.001), whereas a longer TTFT was observed for del(13q) vs. other (131 vs. 51 months, p < 0.0001). Of note, TTFT was not associated with trisomy 12 (p=0.20), trisomy 12 + del(13q) (p=0.61), IgH t(14;18) (p=0.57), normal cytogenetics (p=0.27), or complex cytogenetics (p=0.15). TTFT was similar between patients with mono-allelic, bi-allelic, or mixed del(13q) (131, 103, or 153 months, respectively, p = 0.97). In multivariable analysis, adjusting for ZAP-70 and IGHV, shorter TTFT was independently associated with 14q32 rearrangement (HR 2.51, 95% CI (1.41, 4.45), p=0.002) and del(17p) (HR 1.53, 95% CI (1.0, 2.33), p = 0.047). Del(13q) remained independently associated with longer TTFT (HR 1.97, 95% CI (1.40, 2.77), p < 0.0001). Overall, we show that high risk abnormalities such as del(17p) and del(11q) are associated with unmutated IGHV status and shorter TTFT, and that low risk del(13q) is associated with mutated IGHV, negative ZAP-70 status, and longer TTFT. Also, TTFT is similar between mono- and biallelic del(13q). Interestingly, trisomy 12 was highly associated with unmutated IGHV, but not shorter TTFT, suggesting that trisomy 12 may somehow mitigate the risk of unmutated IGHV. In contrast, 14q32 rearrangement was not associated with either IGHV or ZAP-70, but did predict a short TTFT of 44 months. This suggests that 14q32 rearrangement may hasten the need for treatment, regardless of IGHV or ZAP-70 status. Notably, 14q32 rearrangement due to t(14;18) did not confer increased risk of requiring early therapy. Ongoing analysis will assess overall survival for these cytogenetic abnormalities.Table 1.Cytogenetic abnormalities in 576 CLL patientsCytogenetic Abnormalityn%TTFT (months)del(13q)–total27548131· del(13q x 1)–mono-allelic18667.6% of del(13q)131· del(13q x 0)–bi-allelic238.4% of del(13q)103· del(13q)–mixed5118.5% of del(13q)153Normal701268Trisomy 12631154Trisomy 12 + del(13q)19353Del(17p)47844Del(11q)4172214q32 rearrangement25444IgH t(14;18)19395Complex (3 or more abnormalities)17358 Disclosures: No relevant conflicts of interest to declare.

Abstract 3891 The leukemia B cells of patients (pts) with chronic lymphocytic leukemia (CLL) express a restricted repertoire of immunoglobulin heavy chain variable region (IGHV) genes. Moreover, certain IGHV genes appear over-represented in this repertoire. Among these, IGHV1-69 was first identified as most frequently expressed, being used by the CLL cells of nearly 20% of all pts (PNAS, 86:5913–7, 1989). CLL cells most frequently express IGHV1-69 without somatic mutations and often with restricted D and JH segment use, providing for stereotypic motifs in the heavy chain third complementarity determining region (HCDR3). We addressed whether there were peculiar biologic or clinical features of pts with CLL that used IGHV1-69. For this, we studied 452 pts identified in a cohort of 2,866 followed by the CLL Research Consortium (CRC) found to have CLL cells that express IGHV1-69. This accounted for 16% of all pts. We found that 420 of 452 CLL samples (93%) express IGHV1-69 without somatic mutation (≥98% sequence homology with germline IGHV1-69), which is in significant contrast to the frequency use of UM IGHV among CLL samples that do not use IGHV1-69. As noted for CLL pts in general, there is a strong association between mutation status and clinical behavior. Among pts that use IGHV1-69, the 32 of 452 pts that use MU IGHV1-69 had a highly indolent clinical course, with a median time from diagnosis to initial treatment (TFS) of 17.3 years. This was significantly longer than the median TFS of 2.8 yrs for the 452 pts that used UM IGHV1-69 (p The CLL cells of all 452 pts were examined for ZAP-70. There was a strong association between the expression of ZAP-70 and use of UM IGHV1-69. However, the association between ZAP-70 expression and use of UM IGHV1-69 was not absolute. Only 70% with UM IGHV1-69 were ZAP-70 positive, as were 12.5% that used M IGHV1-69. Of all 452 pts that expressed IGHV1-69, 300 (66%) had ZAP-70 positive CLL cells. These pts had a median TFS of 2.3 yrs, which was significantly shorter than that of the remaining 152 pts with ZAP-70-negative CLL, who had a median TFS of 4.3 yrs (p A Cox regression model revealed that although the presence of detectable chromosomal aberrations was associated to a shorter median TFS, ZAP-70 was a stronger predictor of short TFS (HR for 13q =1.1 p =0.03, HR for trisomy 12 =1.2 p =0.03, HR for 11q =1.6 p =0.03, HR for 17p =1.8, p =0.03) (HR for ZAP-70 positive = 1.8, p=0.0004). The Cox regression model was used to assess the associations of ZAP-70, and the use or not of the UM IGHV1-69 gene with TFS (p-value We conclude that cases that use IGHV1-69 are peculiar in that they more frequently use UM IGHV and appear to have a higher frequency of adverse cytogenetic features than CLL cases at large. In addition, we found that CLL-cell expression of ZAP-70 can segregate pts that use UM IGHV1-69 into subgroups with disparate clinical behavior, despite the fact that all patients use the same IGHV gene. Moreover, multivariable analyses revealed that ZAP-70 was strongest predictor of short TFS among all other considered prognostic parameters in this distinctive cohort of pts. Disclosures: Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Abstract 463 The somatic genetic basis of chronic lymphocytic leukemia (CLL), a common and clinically heterogenous adult leukemia, remains poorly understood. Massively parallel sequencing technology provides a method for systematic discovery of genetic alterations that underlie disease, and for uncovering new therapeutic targets and biomarkers. We therefore performed DNA sequencing of 91 CLL samples with matched germline, consisting of 88 exomes and 3 genomes. Samples were collected from patients displaying a range of characteristics representing the broad clinical spectrum of CLL. Libraries were constructed and sequenced on an Illumina GA-II sequencer to deep coverage. Point mutations and indels were detected by comparing sequences of each tumor to its corresponding normal. We detected 1828 non-silent mutations in protein-coding sequences, corresponding to an average somatic mutation rate of 0.72/Mb (range 0.075–2.14). Nine cancer genes were identified, as they were mutated significantly more often than the background rate given their sequence composition across all 91 CLL/normal pairs. Four have been previously described in CLL (TP53, ATM, MYD88 and NOTCH1); the others were novel (SF3B1, ZMYM3, MAPK1, FBXW7 and DDX3X). Strikingly, SF3B1, which functions at the catalytic core of the spliceosome, was mutated the second most frequently (15%). All 14 identified mutations localized to a discrete region (exons 12–18), and 7 were recurrent (K700E). We additionally validated the SF3B1-K700E mutation through genotyping of 101 independent CLL samples. The 9 significantly mutated genes fell into 5 core signaling pathways, in which the genes play established roles: DNA damage repair and cell-cycle control (TP53, ATM), Notch signaling (FBXW7, NOTCH1), inflammatory pathways (MYD88, DDX3X, MAPK1) and RNA splicing/processing (SF3B1, DDX3X). The common cytogenetic aberrations in CLL carry strong prognostic significance, implying that they reflect distinct pathogenesis. Supporting this hypothesis, we discovered strong associations between different driver mutations and key FISH abnormalities. Most TP53 mutations were in samples with del(17p) (p To define the impact of the mutated cancer genes on disease outcome, we constructed a Cox multivariable regression model for factors contributing to earlier time to first therapy (TTFT) in the 91 CLLs. SF3B1 mutation was predictive of poor prognosis (HR 3.17, p=0.004), independent of other established markers (i.e. IGVH mutation; del(17p); ATM mutation). Consistent with this analysis, patients harboring the SF3B1 mutation alone had short TTFT, as did patients with del(11q) or del(11q)+SF3B1 mutation. All 3 groups demonstrated significantly shorter TTFT than patients without either SF3B1 mutation or del(11q). Because SF3B1 encodes a splicing factor, we looked for functional evidence of splicing alterations associated with SF3B1 mutation. Indeed, mRNA targets of the spliceosome exhibited greater intron retention in CLLs with mutated vs nonmutated SF3B1, confirming alteration in normal SF3B1 function. Understanding the mutational landscape of CLL provides a starting point for systematic analyses to address fundamental questions in CLL, including how mutated genes alter cellular networks and phenotypes, and thereby contribute to disease heterogeneity. Our discovery of striking associations between driver mutations and standard CLL prognostic markers (cytogenetic aberrations, IGVH mutational status) suggest synergistic interactions. In particular, our studies highlight pre-mRNA splicing as a critical but thus far unexplored cellular process contributing to CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 4273 Introduction: Relapsed acute myeloid leukemia (AML) carries a poor prognosis. Although most newly diagnosed patients with AML achieve a complete remission after initial induction (CR1) using standard chemotherapy regimens, relapse follows in the majority of cases. The optimal reinduction chemotherapy regimen for patients following relapse remains unknown, and there is little comparative data to guide selection between various available reinduction regimens. The current study is a single-institution retrospective review over a 10 year period, which seeks to compare rates of response, disease-free survival (DFS) and overall survival (OS) between different reinduction regimens among patients with AML in first relapse. Methods: We performed a retrospective chart review of patients with AML who relapsed following CR1, and who were treated with reinduction chemotherapy at our institution between January 1, 2000, and December 31, 2010. Our patients were categorized into four groups based upon the type of reinduction chemotherapy they received: MEC (mitoxantrone, etoposide, cytarabine) or similar etoposide-containing regimens, 7+3 (infusional cytarabine and anthracycline) or 7+3-based regimens, HiDAC (high-dose cytarabine)-based regimens, and “other” combinations (less frequently used regimens including cytarabine and topotecan; decitabine; gemtuzumab; mitoxantrone and etoposide). Patients were identified using medical billing diagnosis codes, and included if they were above age 18 at diagnosis, obtained CR1 but experienced subsequent relapse, and were treated during first relapse with reinduction with intent to achieve remission. Exclusion criteria included patients with acute promyelocytic leukemia, primary refractory disease, and patients who were given chemotherapy for persistent disease on a mid-treatment bone marrow biopsy. Information about date of diagnosis, treatment regimens, tumor cytopathology and histology, comorbidities at diagnosis, and presenting laboratory data were collected. Statistical analysis was performed using the Kaplan-Meier method, and log rank tests where appropriate, to analyze DFS and OS. Fisher’s exact test was used to assess the associations between categorical variables. Results: We identified 66 patients who were treated with reinduction chemotherapy for AML in first relapse; of these, 28 (42%) achieved CR2. The type of reinduction chemotherapy received was not associated with any difference in the rates of CR2 (p=0.19). Patients who achieved CR2 had a median DFS of 5.1 months. For all patients, including those who failed to achieve CR2, the median OS following reinduction was 4.9 months. There was no significant difference in OS between re-induction regimens (p=0.09). However, there was a statistically significant difference in median DFS depending on regimen (p=0.006). Patients who received 7+3-based regimens, and to a lesser degree MEC, had longer median DFS than HiDAC-based regimens (8.9, 3.4, and 2.0 months, respectively). Patients who received 7+3-based re-induction regimens had a longer duration of preceding CR1 (p=0.006). Longer duration of CR1 (≥7.4 months, the median in our data) was associated with higher rate of CR2 (61% vs 22%, p=0.03) and OS (8.9 vs 2.9 months, p=0.0006) compared to duration of CR1 Conclusion: According to this retrospective analysis, patients treated with MEC or 7+3-based reinduction chemotherapy regimens had improved DFS compared to patients given HiDAC-based regimens. However, as expected, patients treated with 7+3-based reinduction chemotherapy were also more likely to have experienced a longer duration of CR1. Longer CR1 (≥7.4 months) was associated with improved DFS and OS following reinduction chemotherapy. It remains unclear whether the improved DFS seen in those AML patients who received 7+3-based regimens is due to the type of chemotherapy, or if this is related to the characteristics of their underlying disease, as manifested by the longer duration of CR1 seen in this treatment group. Our data does suggest that MEC may be preferable to HiDAC-based regimens for patients with early relapse following CR1. Disclosures: No relevant conflicts of interest to declare.

Abstract 2865 Obatoclax is a small molecule mimetic of the BH3 domain of Bcl-2 family proteins. Obatoclax is broadly specific, with activity against Bcl-2, Bcl-X, Bcl-w and Mcl-1. CLL cells overexpress Bcl-2, Bcl-XL and Mcl-1 in particular, and obatoclax can induce apoptosis of CLL cells in vitro. A phase 1 study of single-agent obatoclax in heavily pretreated largely refractory CLL patients demonstrated that the dose-limiting toxicity was neurologic, including euphoria and ataxia, leading to a maximum tolerated dose of 28 mg/m2 given over 3 hours every 3 weeks. One PR was observed along with biologic activity demonstrated by reductions in lymphocyte counts and improvements in cytopenias. We therefore undertook this phase 1 study of the combination of obatoclax with FR in CLL patients relapsed after at least one prior therapy and in need of therapy again. Obatoclax was given as a three hour infusion on days 1 and 3 at three dose levels, 10, 14, and 20 mg/m2 per dose. Fludarabine was given at 25 mg/m2 days 1–5, and rituximab 375 mg/m2 day 1 following an option to split the dose in cycle 1. Thirteen patients were enrolled, seven men and six women, with median age 58. 5 (38%) had stage 3–4 disease. FISH showed one patient with del17p, one with complex karyotype, and five with del11q. Six of nine patients evaluable had high risk unmutated IGHV, and nine of ten patients evaluable were positive for ZAP-70. The median number of prior therapies was two, with 9 patients having had prior fludarabine-based combination chemotherapy, 10 patients having had prior rituximab, and 8 patients having had prior alkylator-based combination chemotherapy. The study therapy was well-tolerated, with a median of five cycles administered. One dose-limiting toxicity (DLT) was observed at the 20 mg/m2 obatoclax dose; this DLT was a greater than two week treatment delay for persistent grade 2–3 neutropenia in a patient who had had a similar event previously with FR alone. This DLT led to expansion of the third and highest cohort, which enrolled seven patients with no further DLTs observed. Other grade 3–4 toxicities have been limited and include neutropenia (n=5), thrombocytopenia (n=2), fever without neutropenia (n=2), increased ALT/AST (n=1), and dizziness (n=1). Neurologic side effects were easily managed and resembled alcohol intoxication, including grade 1–2 euphoria (n=6), ataxia (n=5), dizziness (n=6), anxiety (n=4), speech impairment (n=4) and confusion (n=3). The ORR by NCI-WG criteria was 85% (11/13; 90% CI 59–97%) with 15% CR (2/13; 90% CI 3–41%) and 38% nPRs (5/13; 90% CI 17–65%). With the addition of CT scan measurement of lymphadenopathy the ORR declined to 54% (7/13; 90% CI 29–78%) with no CRs. With a median follow-up of 26 months from the start of the study, three patients are in ongoing remission, six have relapsed with three receiving further therapy to date, two patients have gone on to stem cell transplantation, and two patients have died of disease. The median time to progression is 20 months (95% CI 9, 35 mos). We were able to demonstrate increased apoptosis compared to baseline in peripheral blood CLL lymphocytes during cycle 1 therapy in 9 of 13 patients, using Annexin V propidium iodide staining. We conclude that the FR-obatoclax regimen is well-tolerated and highly active in a relapsed CLL population. An extension of this study to increase the frequency of obatoclax dosing to days 1–3, and to change the chemotherapy backbone to FCR, is planned pending the availability of obatoclax. Disclosures: Brown: Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding; Pharmacyclics: Consultancy.

Abstract 2846 The immunoglobulin heavy chain variable (IGHV) 1-69 gene, is rearranged in chronic lymphocytic leukemia (CLL) B cells in approximately 20% of the patients, is typically found without somatic mutations, and its expression is often associated with an aggressive clinical course. Unique microRNA (miRs) signatures have been found to be associated with various CLL prognostic markers such as: IGHV mutation status, ZAP-70 expression, and chromosomal aberrations. A karyotype specific signature of some of these miRs has emerged that can differentially segregate patients with adverse clinical behavior and can predict the time from diagnosis to initial treatment. We had interphase FISH available on 281 of the 452 IGHV1-69 cases analyzed in our companion study. We investigated the miRs signature of 53 of these cases that had CLL cells with a sole cytogenetic lesion. All of these cases expressed unmutated (UM) IGHV1-69. These samples included: 18 that had isolated del 11q, 14 that had trisomy 12, 10 that had del 17p and 11 had no detectable chromosomal abnormalities. We did not include any cases that had del 13q. Expression data of the miRs were analyzed by the multiclass comparison within BRB Array Tools to identify a specific set of miRs differentially expressed among the various cytogenetic classes (p=0.05). The data were normalized using the quantiles method. We compared the miRs profiles of CLL cells having defined cytogenetic aberrations with those described in a previously published cohort of 61 CLL patients not selected for IGHV use (Visone et al. Blood 2009). In our cohort of 53 IGHV1-69 cases, we observed significant differences in the expression level of 33 miRs between CLL cells of patients that had no detectable chromosomal aberrations and those of patients with cytogenetic lesions. Of these 2 miRs (miR-543 and miR-769-3p) were published in the cohort of 61 CLL patients. We identified significant differences in the miR expression level of 91 miRs between CLL cells with trisomy 12 and those with other chromosomal aberrations or not. Of these 6 (miR-125b-2*, miR-103, miR-155, miR-107, miR-340 and miR-222) had been previously described in the 61 CLL cohort. We found significant differences in the miR expression level of 26 miRs between CLL cells with the 11q deletion and those with other chromosomal aberrations or not, none of these was previously identified in the 61 CLL cohort. We observed significant differences in the miR expression level of 20 miRs between CLL cells with the 17p deletion and those with other chromosomal aberrations or not, of these only 1 (miR-618) had already been described in the 61 CLL cohort. Since in our companion study on 452 IGHV1-69 cases we found that ZAP-70 expression was a strong predictor of time to first treatment (TFS). We investigated whether CLL expression of ZAP-70 was associated with the peculiar expression of particular miRs in this cohort. Of these 53 patients, 33 (62%) cases had CLL cells that were ZAP-70 positive and had a median TFS of 2.4 years, whereas the 20/53 patients with CLL cells lacking ZAP-70 had a median TFS of 4.0 years. We found that miR-618 and miR-639 were commonly overexpressed in patients with CLL B cells carrying the 11q or 17p deletions and lacking ZAP-70 whereas miR-22 was down-regulated in CLL cells from patients carrying the trisomy 12 or no chromosomal aberrations and lacking ZAP-70. There was a significant difference in miR-618 expression between patients with CLL cells carrying the 11q or 17p chromosomal aberrations with or without ZAP-70 (9.6 vs 10.8, respectively p Disclosures: No relevant conflicts of interest to declare.

Abstract 3555 Introduction: Cytogenetic status represents a major prognostic factor for individuals with AML. However, approximately 50% of individuals have CN-AML and, within this cohort, genetic features have important prognostic and therapeutic implications. Mutations in the FLT3 receptor are found in 30–40% of patients with CN-AML. While the FLT3-internal tandem duplication (ITD) is associated with a poor DFS and OS when compared to CN-AML patients with a wtFLT3R, the prognostic significance of the FLT3-tyrosine kinase domain (TKD) mutation is less clear. There is considerable debate over the best postremission treatment for CN-AML patients who have a FLT3ITD. Our primary objective was to determine the relationship between different postremission therapies and outcome in CN-AML patients with FLT3 mutations. Methods: We retrospectively reviewed the outcomes of newly diagnosed AML patients at the Massachusetts General Hospital and Dana-Farber Cancer Institute/Brigham and Women Hospital between 2002–2008 who were younger than 60 years of age, had a normal karyotype, and had a FLT3ITD and/or TKD mutation at diagnosis. The method of Kaplan and Meier was used to estimate DFS and OS; groups were compared using the log-rank test. A p-value less than .05 was interpreted as statistically significant. Results: At diagnosis, there were 37 patients with an ITD mutation (7 also had a TKD mutation) and 19 patients with an isolated TKD mutation at diagnosis. Patients who received an allogeneic SCT were not included in the analysis. ITD positive patients with an allelic ratio (AR) of mutant to wt FLT3 >.2 had a significantly worse DFS and OS than those with an AR ITD positive patients who underwent an autologous SCT in CR1 had an improved DFS (Figure 1) but similar OS compared to those who received a minimum of three cycles of consolidation chemotherapy (n=8 and 9, respectively) (p=.026 for DFS and .19 for OS). In contrast, for individuals with an isolated TKD mutation, DFS and OS were similar for those who received an autologous SCT (n=6) or consolidation chemotherapy (n=8) (p=.78 for DFS and .25 for OS, respectively). Among patients who initiated postremission therapy, we observed that individuals with an isolated TKD (n=16 for auto SCT or chemotherapy) had an improved DFS and OS compared to ITD patients (n=11) treated with chemotherapy. However, the DFS and OS of TKD patients (n=16 for auto SCT or chemotherapy) versus ITD patients (n=10) who initiated auto SCT was similar (p=.44 for DFS and .09 for OS) (Figure 2A and B, respectively). Conclusion: Our findings suggest that patients with isolated TKD have a similar prognosis regardless of the type of postremission therapy utilized, and that their prognosis is superior to that of patients with ITD. For patients with ITD, autologous SCT in CR1 is associated with improved DFS compared to consolidation chemotherapy alone and is similar to that of patients with TKD. Our data raise the question as to whether autologous SCT should be preferred over consolidation chemotherapy for patients with ITD who are not undergoing allogeneic SCT in CR1 or therapy with a FLT3 inhibitor. Disclosures: DeAngelo: Deminimus: Consultancy. Stone:Novartis: Consultancy.

Abstract 2457 Background: Preclinical studies have shown that CLL cells overexpress Lyn kinase, and in vitro inhibition of Lyn kinase leads to apoptosis of the CLL cells (Contri, J Clin Invest 2005). In 2007, based on this evidence, we initiated a phase II study in patients with previously treated CLL/SLL using dasatinib as a single agent. Preliminary clinical results of this study were reported in 2008. Methods: Patients were required to be over 18 years of age, have a pathologically confirmed diagnosis of CLL/SLL by flow cytometry or immunohistochemistry, and have failed either 1 course of treatment with a fludarabine-containing regimen or 2 non-fludarabine containing regimens. The starting dose of dasatinib was 140 mg daily by mouth. In the first week of therapy sequential blood samples were tested for Lyn kinase protein, p-Lyn, Syk protein, p-Syk, indicators of apoptosis (caspase 3 and Tunel assay), and gene expression profiling among the 3 patients that agreed to participate in this aspect of the study. The final clinical results, with over 1 year follow-up of all patients, showed that among the 15 patients enrolled the major toxicity encountered was myelosuppression: grade 3 + 4 neutropenia in 10 subjects and grade 3 + 4 thrombocytopenia in 6 subjects. Other toxicities included one subject with grade 3 diarrhea, 2 patients with grade 2 pleural effusions, and 1 patient had a transiently prolonged QTc of 516 ms. There were no fatal events, and all toxicities were reversible. In the final analysis partial responses (PR) by NCI-WG criteria were achieved in 3 of the 15 patients (20% with 90% CI 6%-44%). An additional 1 patient would have qualified for an NCI-WG PR except for myelosuppression. Among the 15 patients, 9 had nodal responses (2 CR and 7 PR) by physical exam (PE), 5 without a 50% reduction in lymphocytosis. CT scans confirmed nodal responses in 4 of the 9 patients. Among the 5 patients with del (11q), all 5 had nodal responses by PE. The median time to treatment failure, time to progression, and overall survival were 6.7 months, 7.5 months, and 27 months, respectively. The relationships between clinical response, Lyn kinase, Syk kinase, and apoptosis were analyzed using mononuclear cells from 3 patients at baseline, and at 3, 6, and 24 hours after dosing with dasatinib. For the one of these 3 patients that responded to dasatinib, the high baseline level of p-Lyn was dramatically suppressed at 6 hours correlating with suppression of p-Syk. For the other 2 patients, who did not have a clinical response, there was minimal suppression of p-Lyn and no suppression of p-Syk at 6 hours. Microarray analysis of mRNA from these samples showed that within 6 hours there was significant (p Conclusions: While our in vitro data is very preliminary, it suggests that the activity of dasatinib in CLL may result from the suppression of p-Lyn and inhibition of p-Syk. Disclosures: Amrein: Bristol-Myers Squibb: Honoraria. Off Label Use: Dasatinib used in CLL is off-label use. We show that some patients have a clinical response to the drug in this setting.

Abstract 2689 Introduction: Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by promyelocytes in the blood and bone marrow, coagulopathy, and characteristic translocation between chromosomes 15q22 and 17q21. Internal tandem duplication (ITD) mutations within the FMS-like tyrosine kinase receptor (FLT3) are found in approximately 30% of patients with APL. While cytogenetically normal AML patients with ITD mutations (especially with high ratio of mutant: WT allele) have a poorer prognosis due to high relapse rate, the prognostic value of ITD in APL is debated. We analyzed the clinical outcome of patients with APL to determine the relationship between FLT3 receptor status and prognosis. Materials and Methods: We conducted a retrospective analysis of all adult APL patients < age 60 who were newly diagnosed and tested for FLT3R mutations at Massachusetts General Hospital and Dana-Farber Cancer Institute/Brigham and Women's Hospital between 2002 and 2008. Patient characteristics, complete remission (CR) rates, disease free survival (DFS), and overall survival (OS) were assessed by medical record review under an IRB-approved protocol. CR, DFS and OS were defined according to standard criteria. Kaplan-Meier method was used to estimate median DFS and OS and log-rank p-values were presented. We assessed the associations of clinical characteristics and the type of mutation patients had using Kruskal-Wallis tests. A p-value less than 0.05 was interpreted as statistically significant. Results and Discussion: We identified a total of 26 patients with APL for whom FLT3 mutation status was known. There were 13 patients with wtFLT3, 9 with ITD (1 also had TKD), and the remaining 4 had an isolated TKD mutation. Of the 13 patients with wt FLT3APL, 4 had prior chemotherapy and/or radiation. No patients in the FLT3ITD APL or FLT3TKD APL groups had prior chemotherapy or radiation. As we had only 4 patients had an isolated FLT3TKD mutation, we restricted our analyses to patients who had either wt FLT3 or FLT3ITD+/− TKD. Of the wtFLT3 APL patients, 9/13 were enrolled and treated on CALGB 9710. Of the remaining 4 patients, two were treated according to the PETHEMA regimen, one with a history of breast cancer and significant anthracycline exposure was treated with ATRA and AsO3, and the last patient died before therapy could be initiated. Of the 9 patients with FLT3ITD APL, 5 were treated on CALGB 9710, 2 were treated according to 9710 but off protocol, and 2 with the PETHEMA regimen. Baseline clinical characteristics were similar between the two groups including CR rates [92 (12/13) versus 100% (9/9) for wt and ITD, respectively]. Patients with the ITD had a lower fibrinogen at presentation than those with wt (103 vs. 235 mg/dl, p=.04). While patients with ITD appeared to have a higher WBC, it was not statistically significant (p=.1). However, patients with ITD had an inferior DFS compared to wt (Figure 1) (p=.01) while there was no significant difference in OS between the two groups (p=.39). Of the 5 patients with ITD who relapsed, 3 received AsO3 reinduction and autologous SCT, 1 with CNS recurrence received myeloablative allogeneic SCT, and the fifth died in relapse without treatment. The observation that FLT3ITD APL patients have a reduced DFS raises the possibility that APL therapy may be improved for this group of patients, possibly by incorporating FLT3 inhibitors. Disclosures: DeAngelo: Deminimus: Consultancy. Stone:Novartis: Consultancy.

Abstract 2426 Chronic lymphocytic leukemia is the most common leukemia of adults but still incurable. Prognosis at diagnosis is widely variable, and the key cytogenetic abnormalities determined by FISH remain one of the best predictors of prognosis and treatment response. We therefore undertook very high resolution genomic analysis of 161 CLLs with matched germline samples using Affymetrix 6.0 SNP arrays, in an effort to identify additional predictors of prognosis, and have also performed gene expression profiling on most of this patient cohort. The median age at diagnosis for the cohort was 55 (31–79), and the median time to sampling was 4.6 months (0.5–291). 22% of the cohort was previously treated, with an additional 21% of patients receiving treatment during the follow-up period, for a total of 43% treated, with a median time from diagnosis to treatment of 41 months (0.4-161.2 months). The genomic data were analyzed both by GISTIC, which identifies significant deletions and amplifications based on analysis of the frequency and amplitude of each aberration in the tumor samples alone, as well as by paired copy number analysis of each tumor and its cognate germline, using Birdseed, PLINK and PennCNV. Our results show that the CLL genome is overall quite stable, with a median of only one acquired copy number aberration per sample, excluding rearrangements at the immunoglobulin gene loci. GISTIC analysis on the entire population identified the known common CLL abnormalities at frequencies that would be expected in a largely untreated cohort: 57% del 13q, 6.2% deletion 11q, 5.0% deletion 17p, and 12% trisomy 12. The presence of two or more acquired copy number aberrations (CNAs) of any type was associated with a significantly shorter time to first therapy (p Disclosures: No relevant conflicts of interest to declare.

Abstract 1381 Alemtuzumab is an anti-CD52 antibody originally approved for intravenous administration three times per week to CLL patients refractory to fludarabine and previously exposed to alkylators. Since that time subcutaneous administration three times per week has become widespread because of its reduced infusional toxicity and recently demonstrated equivalent efficacy. In this study we assessed the tolerability, efficacy and pharmacokinetics of administering alemtuzumab subcutaneously weekly at up to 90 mg per dose following an initial 3+3 dose escalation (see table); we further added weekly rituximab in hopes of enhancing activity in lymph nodes. Treatment was administered in up to two eight week blocks with response evaluation between; the second 8 week block continued the dose and schedule used in weeks 5–8. No more than 45 mg was given per subcutaneous injection site. Dose Level Weekly Rituximab Alemtuzumab Weeks 1-2 Alemtuzumab Weeks 3-4 Alemtuzumab Weeks 5-8 Number of Patients 1 375 mg/m2 d 1 30 mg sc d 1, 3, 5 45 mg sc d 1 45 mg sc d 1 3 2 375 mg/m2 d 1 30 mg sc d 1, 3, 5 45 mg sc d 1, 4 45 mg sc d 1 8 3 375 mg/m2 d 1 30 mg sc d 1, 3, 5 45 mg sc d 1, 4 60 mg sc d 1 3 4 375 mg/m2 d 1 30 mg sc d 1, 3, 5 45 mg sc d 1, 4 90 mg sc d 1 14 28 patients were enrolled on this study between 7/2006 and 1/2010. The median age was 62 (range 47–76), and 75% were male. The median time from diagnosis to starting study therapy was 94 mos (14-236 mos). A majority of patients (82%) had Rai stage 3–4 disease and the median number of prior therapies was 4 (1-11). 20/28 patients (71%) had high risk deletions of 17p or 11q. 13/16 (81%) had unmutated IGVH, and 14/19 (74%) were positive for ZAP70. Early study withdrawals occurred due to pre-existing and persistent thrombocytopenia requiring study therapy to be held (n=2), persistent fever attributed to alemtuzumab (n=1), PML in retrospect present prior to study entry (n=1), and a DLT (grade 3 rituximab reaction) which was observed on dose level 2 prior to dose escalation of alemtuzumab. Overall, therapy was well tolerated; injection site reactions were minimal, primarily grade 1 (n=11) with only two grade 2 events. Other toxicities were as expected with alemtuzumab in this patient population, including grade 3–4 neutropenia (54%), grade 3–4 thrombocytopenia (57%), and single cases each of grade 3 rash, AIHA, pulmonary embolism, MRSA bacteremia, diverticular abscess, pulmonary Cryptococcus, EBV lymphoma and metastatic colon cancer. The ORR by NCI-WG criteria at wk 8 was 61% (95% CI 42–76%), with CR rate 11% (95% CI 4–27%). Two of 14 patients who completed a second eight week cycle improved their response (one PR from SD, and one CR from PR). A planned endpoint of this study was to compare lymph node staging by CT to PE, and we found that using CT scans to evaluate nodal response at 8 weeks decreased the ORR rate to 14% (95% CI 6–31%), with no CRs. Bone marrow was completely cleared of disease by 8 weeks in 8 patients and by 16 weeks in an additional 4 patients. The median PFS for the entire population was 13 months with a median follow-up of 9 months in patients who have not progressed. 10 patients have died, 5 of disease, 3 of second malignancies, 1 of PML and 1 of SCT complications. The median OS from study entry is 47 months, with 10 patients having undergone subsequent SCT. Following initiation of therapy we observed a >1,700X decrease in the median CD19+5+ cell count in peripheral blood by the start of week 3. Similar rapid depletion of all T and NK cell subsets was also observed, with first signs of recovery at week 28, and more definite recovery at week 40. Preliminary pharmacokinetic data demonstrated lower maximum levels of rituximab (p=0.06) and alemtuzumab (p=0.05) in patients with >80% bone marrow replacement by CLL but not in those with bulky lymphadenopathy. A trend toward higher alemtuzumab levels was observed in those patients with complete bone marrow clearance (p=0.1) but not in those with objective response. In conclusion, we found that administration of alemtuzumab at 90 mg subcutaneously weekly in combination with rituximab was well-tolerated, convenient and resulted in sustained adequate blood levels of both drugs in most patients. Response rates were high although in this relapsed refractory CLL population, abdominal lymphadenopathy was common, resulting in a decreased response rate when CT scans were included in staging. PFS and OS were favorable for this novel combination regimen and many patients went on to SCT. Disclosures: Brown: Genzyme: Research Funding; Celgene: Consultancy, Research Funding; Calistoga: Consultancy; Genentech: Consultancy. Off Label Use: alternative schedule of alemtuzumab. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

Abstract 59 Immune therapy represents a promising treatment approach in CLL as demonstrated by long-term disease control in pts treated with allogeneic stem cell transplant as well as improved outcomes in pts treated with combination chemoimmunotherapy. Lenalidomide is an immunomodulatory agent with clinical efficacy in CLL. The precise mechanism of lenalidomide's clinical activity in CLL has not yet been defined. Previous reports have demonstrated upregulation of costimulatory molecules on leukemia cells following exposure to lenalidomide and improved capacity of CLL cells to form immune synapses with T cells. Lenalidomide treatment of CLL cells has been associated with an activation phenotype similar to that induced in CD154 gene therapy studies. The CLL Research Consortium is conducting a phase II study to evaluate the combination of lenalidomide and rituximab in treatment-naïve CLL pts who are in need of therapy. This previously untreated population provides a relatively uniform group in which to study the immunomodulatory effects of lenalidomide in CLL. Prior to the administration of rituximab, immunophenotyping was performed on peripheral blood mononuclear cells from pts before and after 21 days of single agent lenalidomide. Mean fluorescent intensity (MFI) was determined on CD19+ lymphocytes relative to isotype-control stained cells. Two-sided Student t-tests were used to assess statistical significance. All pts received lenalidomide 2.5 mg daily for the first 7 days. The dose was escalated to 5 mg on day 8 if they lacked toxicity. 53 pts have been enrolled on study and samples from 38 pts have been assessed for changes in the composition of blood lymphocytes and CLL-cell phenotype following 21 days of single-agent lenalidomide. The absolute numbers of CD19/CD5+ leukemia cells decreased in 35/38 pts (median 48%), whereas the relative percentages of CD4+ and CD8+ T cells increased after therapy. The absolute numbers of T cells increased in 11/38 pts. CLL-cell stained for CD40 had a median MFI of 22 before therapy and 31.1 after therapy (P ≤ 0.0001), increasing in 32/38 pts. Similarly, the median MFI of CLL cells labeled for CD80 or CD86 was respectively 0.7 or 1.8 prior to therapy and 0.9 (p ≤ 0,001) or 3 (P ≤0.02) on day 21. Consistently, the CLL expression of CD54 was increased in post-treatment samples relative to pretreatment CLL cells. The median MFI of CLL cells for CD54 was 54 prior to therapy and 107 on day 21 of therapy (P ≤ 0.00001). Similarly, the median MFI of CD95 (Fas) on CLL cells was 13.1 prior to and 22.5 on day 21 (P ≤ 0.0001). The median MFI of DR5 (CD262) on CLL cells prior to treatment was 10.4 and 15.2 (P ≤ 0.002) post. We observed heterogeneous changes in the expression of HLA-DR on CLL cells; the median MFI of CLL cells for HLA-DR was 189 prior to therapy and 236 (p=0.04) on day 21. We examined whether the relative changes in the CLL cell-surface phenotype were associated with the relative magnitude of the reductions observed in CLL-cell blood counts during the first 21 days of treatment for the first 21 pts studied. We found that leukemia cells from those pts who achieved a > 50% reduction in absolute leukemia cell counts during the first 3 weeks of therapy had significantly greater induction of CD95 (p = 0.01) and HLA-DR (p = 0.004). Similarly, the relative magnitude of increase in expression of CD95 or HLA-DR correlated with the relative reduction in leukemia counts when analyzed as a continuous variable. No significant relationship was observed between the change in CD40 expression and early response to lenalidomide. CD20 expression on CLL cells was not significantly changed from pretreatment levels. Relative increases in the percentage of NK cells observed in these pts may be related to lenalidomide priming of the immune system and contribute to sensitivity to rituximab. Accrual to this study is ongoing. At interim analysis the criteria for continuing accrual to a total of 40 pts in each stratum were met with at least 2 clinical complete responses in each arm. This study reveals a significant association between the relative induction of CD95 and HLA-DR on CLL cells and the relative magnitude of acute reductions observed in leukemia-cell blood counts during the first three weeks of treatment with single-agent lenalidomide. These data are consistent with a model that proposes the relative response to lenalidomide is associated with the relative extent of immune activation of CLL cells induced by lenalidomide in vivo. Disclosures: Off Label Use: lenalidomide in CLL. Wierda:GlaxoSmithKline: Honoraria, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kipps:Genzyme Corporation: Research Funding.

Abstract 3426 Poster Board III-314 Lenalidomide is an immunomodulatory drug recently reported to have an objective response rate (ORR) of 35-53% in relapsed/refractory CLL, even with poor risk features. Initial therapy of CLL currently includes chemoimmunotherapy combinations like FCR, which have high response rates and improved PFS, but at the cost of myelosuppression and infection. Given the high ORR reported with lenalidomide and its potential to spare immune function, we undertook this Phase I study to explore the safety and tolerability of lenalidomide in combination with fludarabine and rituximab. Eligibility criteria included a confirmed diagnosis of CLL/SLL, previously untreated with systemic therapy with an indication for therapy by NCI-WG 1996 criteria; ANC > 1000, platelets > 50K, and adequate organ function. Six dose levels were planned, beginning with fludarabine 25 mg/m2 days 1-3, rituximab 375 mg/m2 day 1, and lenalidomide 2.5 mg administered on days 1-21 of a 28-day cycle. The study used a standard 3+3 dose escalation design, with dose limiting toxicity (DLT) assessed in the first 28 days and defined as grade 3 or greater non-hematologic toxicity, grade 4 neutropenia or thrombocytopenia, grade 3 febrile neutropenia, or a >2-week delay in initiating the second cycle. Nine patients were enrolled on this study, 7 males and 2 females, with a median age of 59 yrs (range 37-66). The median time from diagnosis to study entry was 66.1 months (range 11.7-82.8 months). The median WBC at study entry was 99.6 (range 11.1-325.7). Six patients had Rai 3-4 disease, and three patients had bulky lymphadenopathy (>5 cm) on physical exam. Median beta-2 microglobulin level at study entry was 3.8 mg/L (range 2.5-7.7). The first cohort enrolled four patients, of whom two developed DLTs (grade 4 neutropenia persisting through day 50; febrile syndrome with grade 3 rash, myalgias and grade 4 CPK elevation). The study then proceeded to enroll five patients at dose level -1, with the same dosing of rituximab and fludarabine, but reduced lenalidomide dosing of 2.5 mg every other day for 21 of 28 days. Only two of five patients on dose level -1 completed the planned six cycles of therapy. The other three patients discontinued after 3 cycles due to toxicity: persistent grade 2 thrombocytopenia preventing further therapy; recurrent grade 3-4 neutropenia and thrombocytopenia, despite growth factor support, dose reduction and holding lenalidomide; and intermittent recurrent grade 3 tumor flare, rash and hand-foot syndrome, along with recurrent grade 3 neutropenia despite similar measures as above. At that point the study was closed to enrollment due to the significant myelotoxicity and idiosyncratic tumor flare, resulting in only two of nine patients completing the planned therapy. The median number of cycles completed for all patients was three. Six of the nine enrolled patients were evaluable for response, with five of nine having objective responses (56%, 90% CI 25-83%). No significant difference was observed in IgG and IgM levels between baseline and two months on study, although a trend toward a decrease in IgA levels (p=0.05) was observed. Baseline CD4 counts were variable (median 1139, range 529-2687), but showed significant declines of 574 cells by week 4 (p=0.003) and 707 cells by post-treatment (p=0.002). Analysis of alterations in serum cytokines is ongoing. We conclude that administering lenalidomide concurrently with fludarabine and rituximab is difficult, does not appear to preserve immune function, and limits the ability to deliver adequate therapy with either drug. Other trials currently in progress are exploring alternative schedules of lenalidomide administration with fludarabine and other standard CLL chemotherapy regimens; our data would favor a sequential schedule. Disclosures Brown: Celgene: Honoraria, Provided funding for this investigator-initiated study; Genzyme: Research Funding; Genentech: Consultancy; Calistoga: Consultancy. Off Label Use: Lenalidomide for CLL. Hochberg:Enzon: Speakers Bureau; Biogen-Idec: Speakers Bureau; Genentech: Speakers Bureau; Amgen: Speakers Bureau.